Transgenic animal model and application thereof in simulating human immune system

Abstract

Provided herein are transgenic cells and transgenic non-human animals (e.g., rats and mice) comprising: (i) Rag1 and / or Rag2 gene knockout; (i) knocking out the IL2rg gene; and (iii) a homozygous ineffective mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene, and an optional Fah gene knockout and / or expresses one or more human or humanized polypeptides. Methods and compositions for making and using such transgenic cells and non-human animals are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 390,513, filed on July 19, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Transgenic cells, transgenic mice comprising the transgenic cells, and mice transformed and transplanted, and their use in simulating human diseases, for example, for the purpose of drug testing, are known in the art. It has been reported that transgenic mice are used to simulate the human immune system (HIS) (Manz (2007) Immunity, 26: 537-541). For example, HIS mice produced by transplanting human hematopoietic stem cells and progenitor cells into severe immunodeficient mouse strains (such as Rag2 KOIl2rg KO mice) have been reported. Although multi-lineage hematopoietic development is observed in these HIS mice, it is known that they lack human red blood cells (hRBC) in their peripheral blood. Studies have found that human RBC progenitor cells can develop in the bone marrow of such mice, but cannot survive in the periphery. This hinders the use of these HIS models in simulating diseases or infections associated with human red blood cells and testing the effects of therapeutic drugs on human red blood cells.

[0004] Therefore, there is a need for transgenic mice that can support the maintenance and proliferation of human erythrocytes, as well as mice suitable for implantation that can mimic or approximate certain aspects of human erythrocytes. Summary of the invention

[0005] The present disclosure is based in part on the discovery that knocking out the Hmox-1 gene in immunocompromised mice (eg, mice with Rag1 and / or Rag2 knockouts and IL2rg knockouts) implanted with human hematopoietic cells restores human erythrocytes in peripheral blood.

[0006] Thus, in one aspect, the present disclosure relates to a transgenic non-human animal (e.g., a rodent) comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the transgenic non-human animal comprises a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation in the Hmox-1 gene is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1. In some embodiments, the null mutation in the Hmox-1 gene is a deletion of the entire Hmox-1 endogenous coding sequence.

[0007] In some embodiments, the transgenic non-human animal comprises a homozygous null mutation in the Fah gene. In some embodiments, the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0008] In some embodiments, the transgenic non-human animal expresses a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter. In some embodiments, the transgenic non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 2-4 of a human SIRPA gene. In some embodiments, the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide. In some embodiments, the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene. In some embodiments, the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0009] In some embodiments, the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0010] In some embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters. In some embodiments, an endogenous non-human animal promoter is located at a corresponding non-human animal locus.

[0011] In some embodiments, the transgenic non-human animals described herein comprise a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

[0012] In some embodiments, the transgenic non-human animal is heterozygous for at least one allele, and the allele comprises a nucleic acid sequence encoding a human or humanized protein. In some embodiments, the transgenic non-human animal is homozygous for at least one allele, and the allele comprises a nucleic acid sequence encoding a human or humanized protein.

[0013] In some embodiments, at least one nucleic acid comprises genomic coding and non-coding sequences for a human or humanized protein. In some embodiments, at least one nucleic acid comprises a cDNA sequence for a human or humanized protein.

[0014] In some embodiments, the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0015] In some embodiments, the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter. In some embodiments, the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0016] In some embodiments, the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0017] In some embodiments, the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0018] In certain aspects, the transgenic non-human animals described herein also include an implant of human hematopoietic cells. In some embodiments, the human hematopoietic cells include one or more cells selected from the group consisting of human CD34 positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human erythroid precursor cells, and human erythrocytes. In some embodiments, the animal comprises human erythroid cells.

[0019] In some embodiments, the non-human animal further comprises an infection of a pathogen targeting human erythroid cells. In some embodiments, the animal comprises an inactivated endogenous FAH gene and further comprises transplanted human hepatocytes. In some embodiments, the pathogen can cause human malaria. This pathogen can be selected from Plasmodium sp., Babesia sp., and Theileri sp.

[0020] In some embodiments, the implanted human hematopoietic cells produce abnormal human erythroid cells. In some embodiments, the implanted human hematopoietic cells comprise a mutation in the β-globin gene that causes sickle cell disease.

[0021] In some embodiments, the transgenic non-human animal is a mammal. In some embodiments, the mammal is a rodent, such as a rat or a mouse. In some embodiments, the rodent is a mouse.

[0022] In certain aspects, the present invention provides a method for identifying an agent that inhibits infection by a pathogen targeting human erythroid cells, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; iii. implantation of human hematopoietic cells; and iv. infection by a pathogen targeting human erythroid cells, and b. determining whether the agent reduces the amount of the pathogen and / or inhibits the activity of the pathogen in the non-human animal infected with the pathogen.

[0023] In certain aspects, the present invention provides a method for identifying an agent that prevents infection by a pathogen that targets human erythroid cells, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic cells, b. injecting parasite-infected reticulocytes or erythrocytes into the transgenic non-human animal, and c. determining whether the agent prevents infection of the non-human animal by the human reticulocytes and / or the erythrocytes. In some embodiments, the pathogen can cause human malaria. Such pathogens can be selected from Plasmodium species, Babesia species, and Theileria species.

[0024] In certain aspects, the present invention provides a method for identifying an agent for treating sickle cell disease, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic cells comprising a β-globin gene mutation that causes sickle cell disease, and b. determining whether the agent prevents or reduces sickling of red blood cells in the non-human animal.

[0025] In certain aspects, the present invention provides a method for evaluating the therapeutic effect of a candidate drug targeting human red blood cells, the method comprising: a. administering the candidate drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic progenitor cells, and b. monitoring the human red blood cells in the non-human animal to evaluate the therapeutic effect of the candidate drug.

[0026] In some embodiments, human red blood cells are monitored to determine whether the candidate drug reduces the number of human red blood cells in the non-human animal. In some embodiments, the candidate drug is a chemotherapeutic agent or an anti-malarial agent. In some embodiments, human red blood cells are monitored to assess whether the candidate drug induces agglutination of red blood cells. In some embodiments, the candidate drug is a modulator (e.g., an antibody) of human CD47 protein.

[0027] In certain aspects, the present invention provides a method for identifying an agent that reduces the toxicity of a toxic drug to human red blood cells, comprising: a. administering the agent and the toxic drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic progenitor cells, and b. determining whether the agent reduces the toxicity of the toxic drug to human red blood cells in the non-human animal. In some embodiments, the agent and the toxic drug are administered to the non-human animal simultaneously or sequentially. In some embodiments, the toxicity is on-target toxicity or off-target toxicity.

[0028] In certain aspects, provided herein is a transgenic non-human animal cell comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

[0029] In some embodiments, the transgenic non-human animal cell comprises a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation is a deletion of at least exons corresponding to mouse Hmox-1 exons 3-5. In some embodiments, the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0030] In some embodiments, the transgenic non-human animal cell comprises a homozygous null mutation in the Fah gene. In some embodiments, the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0031] In some embodiments, the transgenic non-human animal cell expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter. In some embodiments, the transgenic non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 2-4 of a human SIRPA gene. In some embodiments, the transgenic non-human animal cell expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide. In some embodiments, the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene. In some embodiments, the transgenic non-human animal cell expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0032] In some embodiments, the transgenic non-human animal cells further express one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0033] In some embodiments, at least one promoter operably connected to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, all promoters operably connected to a nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters. In some embodiments, an endogenous non-human animal promoter is located at a corresponding non-human animal locus.

[0034] In some embodiments, the transgenic non-human animal cell comprises an invalid mutation in at least one corresponding non-human animal gene located at the corresponding non-human animal locus. In some embodiments, the transgenic non-human animal cell is heterozygous for at least one allele, and the allele comprises a nucleic acid sequence encoding the human or humanized protein. In some embodiments, the transgenic non-human animal cell is homozygous for at least one allele, and the allele comprises a nucleic acid sequence encoding the human or humanized protein.

[0035] In some embodiments, the transgenic non-human animal cells express a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0036] In some embodiments, the transgenic non-human animal cells express a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0037] In some embodiments, the transgenic non-human animal cells express a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0038] In some embodiments, the transgenic non-human animal cells express: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0039] In some embodiments, the transgenic non-human animal cells express a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0040] In some embodiments, the transgenic non-human animal cell is a mammalian cell. In some embodiments, the mammalian cell is a rodent cell, such as a rat cell or a mouse cell. In some embodiments, the rodent cell is a mouse cell.

[0041] In some embodiments, the transgenic non-human animal cell is a non-human animal embryonic stem (ES) cell.

[0042] In certain aspects, the present invention provides a method for producing non-human animal embryonic stem cells, comprising genetically engineering the non-human animal embryonic stem cells so that the non-human animal embryonic stem cells have a genome comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0043] In certain aspects, provided herein are non-human animal embryos comprising the non-human animal embryonic stem cells described herein, or non-human animal embryonic stem cells made according to the methods described herein.

[0044] In certain aspects, the present invention provides a method for making a non-human animal, the genome of which contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising the following steps: (a) obtaining the non-human animal embryonic stem cells described herein, or the non-human animal embryonic stem cells made according to the method described herein; and (b) creating a non-human animal using the non-human animal embryonic cells of (a).

[0045] In certain aspects, the present invention provides a method for producing a non-human animal, the genome of which contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising modifying the genome of the non-human animal so that it contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A schematic summary, not shown to scale, of genetic engineering of an exemplary modified Hmox-1 locus according to certain exemplary embodiments provided herein is shown.

[0048] Figure 2A Display HMOX-1 - / - HIS mice showed a decrease in mouse macrophages but not monocytes in the spleen. Specifically, HMOX-1 - / - and HMOX-1 + / +There was no significant difference in mouse CD11b+ / CD14+ monocytes between spleens, but relative to HMOX-1 + / + Spleen, HMOX-1 - / - The F4 / 80+ / VCAM+ macrophage subset was significantly reduced.

[0049] Figure 2B Display HMOX-1 - / - The blood of HIS mice showed a decrease in mouse macrophages, but not monocytes. Although an increase in the CD11b+ / CD14+ monocyte population was observed relative to that of HMOX-1+ / + blood, Figure 2A Similar data were obtained for HMOX-1 - / - The F4 / 80+ macrophage population in the blood was reduced.

[0050] Figure 3 Shown relative to HMOX-1 + / + Mouse, in HMOX-1 - / - The injected human red blood cells (hRBCs) survived longer in the mice.

[0051] Figure 4A HMOX-1 showing engraftment of HSCs - / - HIS mice exhibit hRBCs in peripheral blood.

[0052] Figure 4B HMOX-1 showing engraftment of HSCs - / - and HMOX-1 + / + HIS mice all showed hRBCs in the bone marrow (BM), despite the expression of HMOX-1 - / - The levels of HMOX-1 in HIS mice increased, indicating that although hRBCs can develop in the BM of HIS mice, HMOX-1 - / - HIS mice can allow hRBCs to survive in the peripheral blood.

[0053] Figure 5 Human EPO injection increases HMOX-1 as measured by the percentage of CD235a+ cells - / - hRBC levels in peripheral blood of HIS mice.

[0054] Figure 6 showed that HMOX-1 was significantly increased in both implanted and non-implanted patients, as measured by levels of AST, ALP, ALT, and creatine. - / - Serum chemistry showed no signs of renal / liver damage in HIS mice.

[0055] Figure 7 Display HMOX-1 - / - HIS mice have higher levels of HMOX-1 than HSC donor-matched+ / + HIS mice had a higher implantation rate. Twelve weeks after implantation, HMOX-1 - / - Mice had slightly higher levels of human CD45+, suggesting that the absence of mouse macrophages may have promoted engraftment.

[0056] Figure 8 Display HMOX-1 - / - HIS mice had a decrease in mouse macrophages in the liver, but not monocytes. Since the liver is considered to be the main site for hRBC clearance in mice (Song, Y. et al., Science 371, 1019-1025 (2021)), relative to HMOX-1 + / + , in HMOX-1 - / - A decrease in F4 / 80+ macrophages was observed in the liver of mice, while HMOX-1 - / - The number of total mouse CD11b+ monocytes increased simultaneously. DETAILED DESCRIPTION OF THE INVENTION

[0058] Overview

[0059] The present disclosure relates to transgenic non-human animals (e.g., mice or rats) comprising: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, transgenic non-human animals (e.g., mice) are provided herein, comprising: (i) Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, transgenic non-human animals (e.g., rats) are provided herein, comprising: (i) Rag1 gene knockout; (ii) Rag2 gene knockout; (iii) IL2rg gene knockout; and (iv) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the transgenic non-human animal expresses a human or humanized SIRPA protein encoded by a nucleic acid operably connected to a Sirpa promoter. In some embodiments, the transgenic non-human animal further comprises an inactivation (e.g., deletion) of a fumarylacetoacetase (Fah) gene. In some embodiments, the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably connected to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably connected to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably connected to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably connected to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably connected to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably connected to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably connected to an EPO promoter. In certain embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In other embodiments, transgenic animals express human or humanized nucleic acids from natural human promoters and natural regulatory elements. Professional and technical personnel will understand that transgenic animals include transgenic animals expressing at least one human or humanized nucleic acid from any promoter. Examples of promoters useful in the present invention include, but are not limited to, DNA pol II promoters, PGK promoters, ubiquitin promoters, albumin promoters, globin promoters, ovalbumin promoters, SV40 early promoters, Rous sarcoma virus (RSV) promoters, retroviral LTRs, and lentiviral LTRs. Promoter and enhancer expression systems useful in the present disclosure also include inducible and / or tissue-specific expression systems.

[0060] Rodents, such as mice and rats (e.g., HIS mice and rats) with human immune system components have great prospects for studying the human immune system in vivo and testing human vaccines and testing and developing drugs for treating human diseases and disorders. HIS mice are produced by transplanting human hematopoietic stem cells and progenitor cells (e.g., CD34+HSC) into severe immunodeficient mouse strains (e.g., recombination activation gene 2 (Rag2) knockout (KO) and interleukin 2 receptor gamma (IL2rg) KO mice). Compared with non-human primates, HIS mice have the advantages of small animal models, that is, they allow more versatile experiments, are more easily accepted by the research community, and are more ethically acceptable than conducting experiments with human subjects. Most importantly, the experimental results obtained from HIS mice can be more relevant to humans and applicable to humans. Similar rat models that provide this advantage have also been documented and considered herein.

[0061] Although HIS mice develop human immune cells such as B cells and T cells, human red blood cells (hRBCs) do not develop in the peripheral blood of HIS mouse models. Human RBC progenitor cells can develop in the bone marrow of such mice but cannot survive in the periphery. Therefore, current HIS mice are not well suited for modeling diseases or infections associated with human red blood cells (hRBCs) or for testing the effects of therapeutic drugs on human RBCs.

[0062] To overcome these limitations, the present disclosure provides an Hmox-1-deficient HIS mouse model. Infused human red blood cells are rapidly cleared from mice, and their destruction is primarily thought to be due to phagocytosis by mouse macrophages. This idea is supported by the fact that elimination of macrophages with in vivo clodronate liposome treatment prolongs the survival of human red blood cells. However, this approach requires repeated injections, which is not feasible due to mouse toxicity. Hmox-1 deficiency in mice leads to a loss of erythrophagic macrophages (i.e., macrophages that eat red blood cells). This is because the Hmox-1 enzyme is required for macrophages to break down and recycle heme in engulfed red blood cells. With the loss of this enzyme, toxic heme accumulates in erythrophagic macrophages, leading to their death. Therefore, Hmox-1KO in the HIS model allows human red blood cells to survive after HSC implantation. This article demonstrates that Hmox-1-deficient hSIRPA Rag2 mice are significantly more resistant to HSC transplantation than HSC donor-matched Hmox-1 competent control mice. - / - IL-2Rγ - / - The mice exhibit human red blood cells in their peripheral blood. This new model provides a useful tool for studying human red blood cell biology and also provides a useful model for developing treatments for red blood cell diseases.

[0063] Transgenic non-human animals provided herein have many uses in the art, including, for example, for simulating human erythropoiesis and erythrocyte function; simulating human pathogen infection of erythrocytes; in vivo screening of agents that regulate erythropoiesis and / or erythrocyte function, for example, in a healthy or diseased state; in vivo screening of agents that are toxic to erythrocytes or erythrocyte progenitors; in vivo screening of agents that prevent, reduce or reverse the toxic effects of toxic drugs on erythrocytes or erythrocyte progenitors; in vivo screening of erythrocytes or erythrocyte progenitors from individuals to predict the responsiveness of individuals to disease therapies. These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods described more fully below.

[0064] definition

[0065] As used herein, the articles "a" and "an" refer to one or more (ie, to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or a plurality of elements.

[0066] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both an amino functional group and an acid functional group and that are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0067] The "coding region" of a gene includes nucleotide residues of the gene coding chain and nucleotides of the non-coding chain of the gene, which are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription, respectively. The "coding region" of an mRNA molecule also includes nucleotide residues of the mRNA molecule, which match or encode the termination codon with the anticodon region of the transfer RNA molecule during the translation of the mRNA molecule. Therefore, the coding region can include nucleotide residues of codons containing amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in the protein export signal sequence).

[0068] As used herein, the phrase "endogenous gene" or "endogenous gene fragment" refers to a gene or gene fragment found in a parent or reference organism prior to the introduction of a disruption, deletion, substitution, alteration, or modification as described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-grown organism (whether wild-type or engineered).

[0069] As used herein, the term "chimera" refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) includes parts from different species. In some embodiments, a "chimeric" nucleic acid or protein described herein includes nucleotides or amino acid sequences from both non-human sources and from humans. In such embodiments, a "chimeric" nucleic acid or protein may also be referred to as a "humanized" nucleic acid or protein.

[0070] As used herein, the term "corresponding to" refers to exons encoding the same or homologous functional domains or protein portions. For example, "exons corresponding to mouse Hmox-1 exons 3-5" refers to exons from a transgenic non-human animal that encode the same or homologous functional domains or protein portions as mouse exons 3-5. These may be exons 3-5 of the transgenic non-human animal, or other exons due to different exon configurations between different non-human animal species.

[0071] The term "humanization" refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) includes a portion from a non-human source, which is engineered to have a structure and function more similar to a real human nucleic acid or protein than the nucleic acid or protein of the original source. For example, humanization can involve selecting amino acid substitutions to make the non-human sequence more similar to the human sequence. Humanization can also involve transplanting at least a portion of a non-human protein into a human protein. To give just one example, in the case of a membrane receptor, a "humanized" gene can encode a polypeptide whose extracellular portion has the same amino acid sequence as the human extracellular portion, while the remaining sequence is the same as the amino acid sequence of a non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene comprises at least a portion of a DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence having a portion that appears in a human protein. The term "human" is recognized in the field and refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) is completely from a human source.

[0072] As used herein, the term "locus" refers to a position on a chromosome that contains a group of related genetic elements (e.g., genes, gene fragments, regulatory elements). The locus can be endogenous or non-endogenous. The term "endogenous locus" refers to the position of a specific genetic element naturally present on a chromosome. In some embodiments, the endogenous locus has a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the endogenous locus is an engineered locus.

[0073] As used herein, the phrase "non-human animal" refers to any non-human vertebrate organism. In some embodiments, the non-human animal is a cyclostomum vertebrate, a bony fish, a cartilaginous fish (e.g., a shark or ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent, such as a rat or a mouse.

[0074] As used herein, the phrase "operably linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in the intended manner. A control sequence that is "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or remotely to control a gene of interest. As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0075] The terms "polynucleotide" and "nucleic acid" can be used interchangeably. They refer to the polymer form of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding regions or non-coding regions of genes or gene fragments, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. Polynucleotides can be further modified, such as by being conjugated with a labeling component. In all nucleic acid sequences provided herein, U nucleotides can be interchanged with T nucleotides.

[0076] As used herein, the term "polypeptide" refers to any polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, the polypeptide has an engineered amino acid sequence because it is designed and / or produced by artificial action.

[0077] As used herein, the term "promoter" includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. The promoter is usually located upstream of the nucleic acid sequence to be transcribed, and provides a site for the specific binding of RNA polymerase and other transcription factors. In a specific embodiment, the promoter is usually located upstream of the transcribed nucleic acid sequence to produce the desired molecule, and provides a site for the specific binding of RNA polymerase and other transcription factors. The phrase "endogenous promoter" refers to a promoter that is naturally associated with an endogenous gene, for example, in a wild-type organism.

[0078] As used herein, the term "recombinant" refers to a polypeptide (e.g., a signal regulating protein as described herein) that is designed, engineered, prepared, expressed, created, or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library (Hoogenboom HR, (1997) TIBTech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), an antibody isolated from an animal (e.g., mouse) transgenic for a human immunoglobulin gene (see, e.g., Taylor, LD, et al., (1992) Nucl. Acids Res.20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597, Little M. et al., (2000) Immunology Today 21:364-370) or prepared, expressed, created or isolated polypeptides by any other means, including splicing selected sequence elements to each other. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed by computer. In some embodiments, one or more of one or more such selected sequence elements are produced by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, for example, from natural or synthetic sources. For example, in some embodiments, the recombinant polypeptide consists of a sequence found in the genome of an organism of interest (e.g., human, mouse, etc.). In some embodiments, a recombinant polypeptide has an amino acid sequence that results from mutagenesis (e.g., in vitro or in vivo, such as in a non-human animal), such that the amino acid sequence of the recombinant polypeptide is a sequence that, while derived from and related to the polypeptide sequence, may not naturally exist in the genome of the non-human animal in vivo.

[0079] As used herein, the term "replacement" refers to the process in which a "replaced" nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from the locus and a different, "replaced" nucleic acid is positioned in its place. In some embodiments, the replaced nucleic acid sequence and the replaced nucleic acid sequence are comparable to each other, for example, they are homologous to each other and / or contain corresponding elements (e.g., protein coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice receiving site, an intron, an exon, and an untranslated region (UTR); in some embodiments, the replaced nucleic acid sequence includes one or more coding sequences. In some embodiments, the replaced nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replaced nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, the replaced nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the following situations are included: wherein the replaced nucleic acid sequence is or comprises a human nucleic acid sequence, and the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse sequence). The nucleic acid sequence so arranged may include one or more regulatory sequences that are part of the source nucleic acid sequence used to obtain the sequence so arranged (e.g., promoter, enhancer, 5'- or 3'-untranslated region, etc.). For example, in various embodiments, the replacement is the replacement of the endogenous sequence with a heterologous sequence, which results in the production of a gene product from the nucleic acid sequence so arranged (including the heterologous sequence), but does not produce the expression of the endogenous sequence; the replacement is the replacement of the endogenous genomic sequence with a nucleic acid sequence, and the protein encoded by the nucleic acid sequence has a similar function to the protein encoded by the endogenous sequence. In various embodiments, the endogenous gene or fragment thereof is replaced by the corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is an ortholog of the replaced endogenous gene or fragment, and is a human gene or fragment that is substantially similar or identical in structure and / or function to the replaced endogenous gene or fragment.

[0080] As used herein, the term "variant" includes such nucleic acid sequences or peptide sequences, whose sequences are different from the reference nucleic acid sequence or peptide sequence, respectively, but retain the basic biological properties of the reference molecule. The changes in the nucleic acid variant sequence may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may cause amino acid substitutions, additions, deletions, fusions and truncations. The changes in the peptide variant sequence are usually limited or conservative, so the sequences of the reference peptide and the variant are generally very similar and identical in many regions. Variants and reference peptides can be distinguished by any combination of one or more substitutions, additions, and deletions in the amino acid sequence. The variant of a nucleic acid or peptide can be naturally occurring, such as an allelic variant, or can be an unknown naturally occurring variant. Non-naturally occurring variants of nucleic acids and peptides can be prepared by mutagenesis techniques or direct synthesis.

[0081] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid associated with it. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in host cells, such as eukaryotic cells and / or prokaryotic cells. Vectors capable of directing the expression of operably linked genes are referred to herein as "expression vectors."

[0082] As used herein, the term "wild-type" has its art-understood meaning and refers to an entity having a structure and / or activity found in nature in a "normal" (as opposed to mutated, diseased, altered, etc.) state or background. One of ordinary skill in the art will recognize that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).

[0083] Transgenic locus

[0084] In certain aspects, provided herein are transgenic non-human animals (e.g., mice or rats) comprising: (i) Rag1 and / or Rag2 gene knockouts; (ii) IL2rg gene knockouts; and (iii) homozygous null mutations in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, provided herein are transgenic non-human animals (e.g., mice) comprising: (i) Rag2 gene knockouts; (ii) IL2rg gene knockouts; and (iii) homozygous null mutations in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, provided herein are transgenic non-human animals (e.g., rats) comprising: (i) Rag1 gene knockouts; (ii) Rag2 gene knockouts; (iii) IL2rg gene knockouts; and (iv) homozygous null mutations in the Hmox-1 gene. In some embodiments, transgenic non-human animals express human or humanized SIRPA proteins encoded by nucleic acids operably linked to a Sirpa promoter. In certain embodiments, the transgenic non-human animal also expresses one or more human or humanized proteins selected from the group consisting of: human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and human EPO protein encoded by a nucleic acid operably linked to an EPO promoter. In some embodiments, the transgenic non-human animal further comprises inactivation (e.g., deletion) of a fumarylacetoacetase (FAH) gene. In certain embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, the transgenic non-human animal comprises implantation of human hematopoietic stem cells (HSCs). In some embodiments, the transgenic non-human animal comprises an implantation of human hepatocytes.

[0085] Hmox-1 knockout

[0086] In certain aspects, the transgenic non-human animals (eg, mice or rats) provided herein comprise a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0087] Heme oxygenase metabolizes heme and releases free iron, carbon monoxide, and biliverdin, which is rapidly converted to bilirubin. Humans and mice contain two well-characterized heme oxygenases: HMOX-1, which is inducible, and HMOX-2, which is constitutively expressed in most tissues (Kovtunovych et al., (2010) Blood 116: 6054-6062). Previous studies in mouse models have shown that the lack of both Hmox-1 and Hmox-2 is embryonic lethal. A human patient with HMOX-1 deficiency died at the age of 6 years, showing growth retardation, severe anemia, endothelial cell damage, spleen, liver, kidney fibrosis, and liver and kidney iron overload (Kawashima et al., (2002) Hum. Path. 33: 125-130; Yachie et al., (1999) J. Clin. Invest. 103: 129-135). Hmox-1 - / - Mice are anemic, but oxidatively stressed erythrocytes live longer. - / - Splenomegaly caused by increased red pulp was observed in mice, and splenic fibrosis was observed in aged mice. - / - Iron accumulation in the kidneys of mice leads to kidney damage in these mice. - / - Macrophage deficiency in the spleen and liver has also been observed in mice (Poss et al., (1997) PNAS 94:10919-10924; Kovtunovych et al., (2010) Blood 116:6054-6062; Kovtunovych et al., (2014) Blood 124:1522-1530). For example, Hmox-1 - / - Mice had reduced macrophages (F4 / 80+) in the spleen, liver, blood, and bone marrow (BM), but still had myeloid cells (CD11b + ) (Fraser et al., (2015) Haematologica 100:601-610). Lack of Hmox-1 renders hemophagocytic macrophages unable to process heme and causes intracellular toxicity due to heme accumulation (Kovtunovych et al., (2010) Blood 116:6054-6062). Wild-type bone marrow transplantation reverses Hmox-1 - / - Disease in mice is a result of restoration of the heme cycle by repopulating tissues with wild-type macrophages (Kovtunovych et al. (2014) Blood 124:1522-1530).

[0088] Representative human HMOX-1 cDNA and human HMOX-1 protein sequences are known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, human (NP_002124.1) can be encoded by transcript (NM_002133.3). The nucleic acid and polypeptide sequences of Hmox-1 orthologs in organisms other than humans are well known, including, for example, chimpanzee Hmox-1 (XM_525579.6 and XP_525579.2), rhesus monkey Hmox-1 (XM_028827760.1 and XP_028683593.1), bovine Hmox-1 (NM_001014912.1 and NP_001014912.1), canine Hmox-1 (NM_001194969.1 and NP_001181898.1), rat Hmox-1 (NM_012580 .2 and NP_036712.1), mouse Hmox-1 (NM_010442.2 and NP_034572.1), Chinese hamster Hmox-1 (XM_003511957.5 and XP_003512005.1), chicken Hmox-1 (XM_046921508.1 and XP_046777464.1), tropical clawed frog Hmox-1 (XM_002934720.5 and XP_002934766.2), and zebrafish Hmox-1 (NM_001127516.1 and NP_001120988.1).

[0089] In certain aspects, the transgenic non-human animal comprises a homozygous null mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene. Null mutations include deletions, insertions and / or substitutions in a gene, which result in a non-functional gene product (e.g., a complete lack of a gene product (protein, RNA) at a molecular level, or expression of a non-functional gene product). In the present disclosure, null mutations have the same meaning as inactivating mutations and can be used interchangeably with inactivating mutations. Homozygous null mutations refer to null mutations in all alleles. For example, a homozygous null mutation in a mouse or rat Hmox-1 gene refers to a null mutation in two alleles of a mouse or rat Hmox-1 gene (i.e., two null alleles). Genes with homozygous null mutations are also referred to as knockouts or gene defects / gene defects. For example, homozygous null mutations in Hmox-1 are also referred to as Hmox-1 knockouts or Hmox-1 defects. Therefore, null mutations in non-human animal Hmox-1 genes include deletions, insertions and / or substitutions in non-human animal Hmox-1 genes. In some cases, the endogenous non-human animal Hmox-1 locus comprises a null mutation, and therefore comprises a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of the gene. This can be due to the complete lack of expression of a gene product (protein, RNA) or a non-functional gene product at the molecular level. At the phenotypic level, a null allele comprises the disappearance of the entire locus.

[0090] In some embodiments, the null mutation is a deletion of at least exons corresponding to mouse Hmox-1 exons 3-5. In some embodiments, the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence. In some embodiments, the non-human animals provided herein do not express Hmox-1 protein.

[0091] In some embodiments, the homozygous null mutation in the Hmox-1 gene of a non-human animal comprises the same null mutation for all alleles. In some embodiments, the homozygous null mutation in the Hmox-1 gene of a non-human animal comprises different null mutations for different alleles.

[0092] Mouse Hmox-1 is located on chromosome 8, GRCm39, NC_000074.7 (75820246-75827221), and the coding sequence of mouse Hmox-1 can be found in Genbank accession number NM_010442.2. The mouse Hmox-1 locus includes 5 exons, and exons 1-5 are coding exons. Therefore, in some embodiments, the transgenic animals provided herein are mice, and one or more of exons 1-5 of the mouse Hmox-1 gene are deleted or mutated in the transgenic mice. In some cases, other aspects of the genomic site of the mouse Hmox-1 gene, such as introns, 3' and / or 5' untranslated sequences (UTRs), are also deleted or mutated. In some cases, the entire region of the mouse Hmox-1 genomic site is deleted. In some embodiments, the entire genomic region from the start codon to the stop codon of the mouse Hmox-1 gene is deleted. For example, a transgenic mouse can contain a deletion of ~7 kb mouse sequence (GRCm38 coordinates chr8: 75093750-75100019), as shown in Example 1.

[0093] A variety of methods can be used to detect a deleted, modified or altered Hmox-1 gene at the endogenous Hmox-1 locus, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or gain or loss of alleles assay. In some embodiments, the non-human animal is homozygous for a deletion or null mutation of the endogenous Hmox-1 gene.

[0094] In some embodiments, a non-human animal (e.g., mouse or rat) comprising a homozygous null mutation of the Hmox-1 gene, i.e., a non-human animal with Hmox-1 deficiency, is an immunocompromised animal. For example, a non-human animal (e.g., mouse or rat) with Hmox-1 deficiency may include at least one null allele of the Rag2 gene ("recombination activating gene 2", wherein the coding sequence of the mouse gene can be found in Genbank accession number NM_009020.3). In some embodiments, a non-human animal (e.g., mouse or rat) with Hmox-1 deficiency includes two null alleles of Rag2. In other words, a non-human animal (e.g., mouse or rat) with Hmox-1 deficiency is homozygous null for Rag2. In other embodiments, a non-human animal (e.g., mouse or rat) with Hmox-1 deficiency includes one or two null alleles of the Rag1 gene. In some embodiments, a non-human animal (e.g., mouse or rat) with Hmox-1 deficiency is homozygous null for Rag1. In some embodiments, the non-human animal (e.g., mouse or rat) deficient in Hmox-1 includes (i) one or two null alleles of the Rag1 gene; and (ii) one or two null alleles of the Rag2 gene. In some embodiments, the non-human animal (e.g., mouse or rat) deficient in Hmox-1 is homozygous null for both Rag1 and Rag2. In some embodiments, the non-human animal deficient in Hmox-1 is an immunocompromised mouse, comprising two null alleles of Rag2 (i.e., homozygous null). In some embodiments, the non-human animal deficient in Hmox-1 is an immunocompromised rat, comprising two null alleles of Rag1 (i.e., homozygous null) and two null alleles of Rag2 (i.e., homozygous null). As another example, a non-human animal (e.g., a mouse or rat) deficient in Hmox-1 includes at least one null allele of the IL2rg gene ("interleukin 2 receptor, gamma," also known as common gamma chain, or gamma C, wherein the coding sequence of the mouse gene can be found in Genbank Accession No. NM 013563.4). In some embodiments, a non-human animal (e.g., a mouse or rat) deficient in Hmox-1 includes two null alleles of IL2rg. In other words, a non-human animal (e.g., a mouse or rat) deficient in Hmox-1 is homozygous null for IL2rg, i.e., it is IL2rg - / - (or IL2rg Y / - , wherein the IL2rg gene is located on chromosome X of the mouse). In some embodiments, the Hmox-1-deficient non-human animal (eg, mouse or rat) comprises a null allele of both Rag2 and IL2rg, i.e., it is Rag2 - / - IL - / - (or Rag2 - / -IL Y / - , wherein the IL2rg genes in mice or rats are all located on chromosome X). In some embodiments, the non-human animal (e.g., mouse or rat) deficient in Hmox-1 comprises null alleles of Rag1 and IL2rg. In some embodiments, the non-human animal (e.g., mouse or rat) deficient in Hmox-1 comprises null alleles of Rag1, Rag2, and IL2rg. In some embodiments, the non-human animal deficient in Hmox-1 is an immunocompromised mouse, comprising two null alleles of Rag2 (i.e., homozygous null) and two null alleles of IL2rg (i.e., homozygous null) (or one null allele for male mice). In some embodiments, the non-human animal deficient in Hmox-1 is an immunocompromised rat, comprising two null alleles of Rag1 (i.e., homozygous null), two null alleles of Rag2 (i.e., homozygous null), and two null alleles of IL2rg (i.e., homozygous null) (or one null allele for male rats). Other genetic modifications are also contemplated. For example, Hmox-1-deficient non-human animals (e.g., mice or rats) may include modifications of other genes related to the development and / or function of hematopoietic cells and the immune system, for example, replacing one or more other non-human animal genes with nucleic acid sequences encoding human or humanized polypeptides. Such genes include, but are not limited to, for example, SIRPA, CD47, M-CSF, GM-CSF, TPO, EPO, IL-3, and IL-15. Additionally or alternatively, Hmox-1-deficient non-human animals (e.g., mice or rats) may include modifications of genes related to the development and / or function of other cells and tissues, for example, genes related to human conditions or diseases, or genes that provide human conditions and disease models when modified in non-human animals (e.g., mice). In one embodiment described below, Hmox-1-deficient non-human animals (e.g., mice or rats) may include mutations, insertions, or deletions in the Fah gene. The introduction of other genetic modifications may be accomplished by ES cell modification and / or breeding. For example, Hmox-1-deficient (and optionally Rag2 and IL2rg-deficient) non-human animals (e.g., mice or rats) can be crossbred with non-human animals comprising one or more other genetic modifications, including, but not limited to, for example, modifications of SIRPA, CD47, M-CSF, GM-CSF, TPO, EPO, IL-3, IL-15, and / or Fah genes. In some embodiments, all genetic modifications are bred to homozygosity in the transgenic animals described herein.

[0095] Immunodeficient non-human animals

[0096] As described above, transgenic non-human animals comprising the Hmox-1 deficiency described herein are also immunodeficient because they comprise deficiencies in the Rag1 and / or Rag2 and Il2rg genes. Rag1, Rag2, and Il2rg are important components of the adaptive immune system. When one or more of these genes mutate in an animal, T cells and B cells do not mature and the animal is severely impaired. When these animals are attacked by xenotransplanted cells, they are unable to mount an immune response to the foreign cells.

[0097] V(D)J recombination activating protein 1 (also known as RAG1, recombination activation 1, recombination activation gene 1, and recombination activating protein 1) is encoded by the Rag1 gene (also known as recombination activation 1). RAG1 is the catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles a diverse repertoire of immunoglobulin and T cell receptor genes in developing B and T lymphocytes by rearranging different V (variable), and in some cases D (diversity), and J (joining) gene segments. Within the RAG complex, RAG1 mediates DNA binding to a conserved recombination signal sequence (RSS) and catalyzes DNA cleavage activity by introducing double-strand breaks between the RSS and adjacent coding segments. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential for the generation of mature B cells and T cells, two types of lymphocytes that are key components of the adaptive immune system.

[0098] Mouse Rag1 is located at 2E2 on chromosome 2; 2 53.88 cM (NCBI RefSeq Gene ID 19373; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101468597..101479877, complement). Reference to the mouse Rag1 gene, including the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type mouse RAG1 protein is assigned UniProt accession number P15919 and NCBI accession number NP_033045.2. Reference to the mouse RAG1 protein, including the wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform is assigned NCBI accession number NM_009019.2. Reference to the mouse Rag1 mRNA (cDNA) and coding sequence, including the classical wild-type form as well as all allelic forms and isoforms.

[0099] Rat Rag1 is located at 3q31 on chromosome 3 (NCBI RefSeq Gene ID 84600; assembly mRatBN7.2 (GCF_015227675.2); position NC_051338.1 (87917061..87928158, complement). References to the rat Rag1 gene include the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type rat RAG1 protein has been assigned UniProt accession number G3V6K9 and NCBI accession number NP_445920.1. References to rat RAG1 proteins include the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform has been assigned NCBI accession number NM_053468.1. References to rat Rag1 mRNA (cDNA) and coding sequences include the classical wild-type form as well as all allelic forms and isoforms.

[0100] An inactivated endogenous Rag1 gene is a Rag1 gene that does not produce RAG1 protein or does not produce functional RAG1 protein. A non-human animal (or cell or genome) may contain an inactivated Rag1 gene in its germline. The non-human animal (or cell or genome) may be homozygous for an inactivating mutation in the Rag1 gene. As an example, an inactivated endogenous Rag1 gene may comprise an insertion, deletion, or one or more point mutations in the endogenous Rag1 gene, resulting in a lack of expression of functional RAG1 protein. Some inactivated endogenous Rag1 genes may include the deletion or destruction of all endogenous Rag1 genes, or may include the deletion or destruction of an endogenous Rag1 gene fragment (i.e., a portion of a gene or a portion of a gene). For example, some, most, or all of the coding sequences in the endogenous Rag1 gene may be deleted or destroyed. In one example, the 5' fragment of the Rag1 gene may be deleted or destroyed (e.g., including a start codon). As an example, an inactivated endogenous Rag1 gene can be a gene in which the start codon of the endogenous Rag1 gene has been deleted or destroyed or mutated so that the start codon no longer functions. For example, the start codon can be destroyed by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, replacing one or more nucleotides. In another example, the 3' fragment of the Rag1 gene can be deleted or destroyed (e.g., including a stop codon). In another example, an internal fragment of the Rag1 gene (i.e., a fragment from the middle of the Rag1 gene) can be deleted or destroyed. In another example, all coding sequences in the endogenous Rag1 gene are deleted or destroyed.

[0101] V(D)J recombination activating protein 2 (also known as RAG2, recombination activation 2, recombination activation gene 2, and recombination activating protein 2) is encoded by the Rag2 gene (also known as recombination activation 2). As mentioned above, RAG1 is the catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential for the generation of mature B cells and T cells, two types of lymphocytes that are key components of the adaptive immune system.

[0102] Mouse Rag2 is located at 2E2 on chromosome 2; 2 53.87 cM (NCBI RefSeq Gene ID 19374; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101455057..101462873). Reference to the mouse Rag2 gene, including the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type mouse RAG2 protein has been assigned UniProt accession number P21784 and NCBI accession number NP_033046.1. Reference to the mouse RAG2 protein, including the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform is assigned NCBI accession number NM_009020.3. Reference to the mouse Rag2 mRNA (cDNA) and coding sequence, including the classical wild-type form as well as all allelic forms and isoforms.

[0103] Rat Rag2 is located at 3q31 on chromosome 3 (NCBI RefSeq Gene ID 295953; assembly mRatBN7.2 (GCF_015227675.2); position NC_051338.1 (87902373..87910227). Reference to rat Rag2 gene, including the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type rat RAG2 protein has been assigned UniProt accession number G3V6K7 and NCBI accession number NP_001093998.1. Reference to rat RAG2 protein, including the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform is assigned NCBI accession number NM_001100528.1. Reference to rat Rag2 mRNA (cDNA) and coding sequence, including the classical wild-type form as well as all allelic forms and isoforms.

[0104] An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce RAG2 protein or does not produce functional RAG2 protein. A non-human animal (or cell or genome) may contain an inactivated Rag2 gene in its germline. A non-human animal (or cell or genome) may be homozygous for an inactivating mutation in a Rag2 gene. As an example, an inactivated endogenous Rag2 gene may comprise an insertion, deletion, or one or more point mutations in an endogenous Rag2 gene, resulting in loss of expression of a functional RAG2 protein. Some inactivated endogenous Rag2 genes may include the deletion or destruction of all endogenous Rag2 genes, or may include the deletion or destruction of an endogenous Rag2 gene fragment (i.e., a portion of a gene or a portion of a gene). For example, some, most, or all of the coding sequences in an endogenous Rag2 gene may be deleted or destroyed. In one example, the 5' fragment of the Rag2 gene may be deleted or destroyed (e.g., including a start codon). As an example, an inactivated endogenous Rag2 gene can be a gene in which the start codon of the endogenous Rag2 gene has been deleted or destroyed or mutated so that the start codon no longer functions. For example, the start codon can be destroyed by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, replacing one or more nucleotides. In another example, the 3' fragment of the Rag2 gene can be deleted or destroyed (e.g., including a stop codon). In another example, an internal fragment of the Rag2 gene (i.e., a fragment from the middle of the Rag2 gene) can be deleted or destroyed. In another example, all coding sequences in the endogenous Rag2 gene are deleted or destroyed.

[0105] Interleukin 2 receptor subunit gamma (also known as interleukin 2 receptor, gamma; interleukin 2 receptor, gamma (severe combined immunodeficiency), isoform CRA_a; cytokine receptor common subunit gamma precursor) is encoded by the Il2rg gene (also known as interleukin 2 receptor subunit gamma or IL2RG). IL2RG is a cytokine receptor subunit that is common to the receptor complexes of several different interleukin receptors. IL2RG is located on the surface of immature hematopoietic cells in the bone marrow. IL2RG works with other proteins to direct the formation of lymphocytes from hematopoietic cells. IL2RG also directs the growth and maturation of T cells, B cells, and natural killer cells. Mutations in Il2rg can cause X-linked severe combined immunodeficiency, in which lymphocytes do not develop normally. The lack of functional mature lymphocytes impairs the ability of the immune system to protect the body from infection.

[0106] Mouse Il2rg is mapped to XD on chromosome X; X 43.9 cM (NCBI RefSeq Gene ID 16186; assembly GRCm39 (GCF_000001635.27); position NC_000086.8 (100307991..100311861, complement). References to the mouse Il2rg gene include the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type mouse IL2rG protein has been assigned UniProt accession number P34902 and NCBI accession number NP_038591.1. References to the mouse IL2RG protein include the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform has been assigned NCBI accession number NM_013563.4. References to mouse Il2rg mRNA (cDNA) and coding sequences, including the classical wild-type form as well as all allelic forms and isoforms.

[0107] Rat Il2rg is located at Xq22 on chromosome X (NCBI RefSeq Gene ID 140924; assembly mRatBN7.2 (GCF_015227675.2); position NC_051356.1 (66395330..66399026, complement). References to the rat Il2rg gene include the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type rat IL2rG protein has been assigned UniProt accession number Q68FU6 and NCBI accession number NP_543165.1. References to the rat IL2RG protein include the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform has been assigned NCBI accession number NM_080889.1. References to the rat Il2rg mRNA (cDNA) and coding sequences, including the classical wild-type form as well as all allelic forms and isoforms.

[0108] An inactivated endogenous Il2rg gene is an Il2rg gene that does not produce IL2RG protein or does not produce functional IL2RG protein. A non-human animal (or cell or genome) may contain an inactivated Il2rg gene in its germline. The non-human animal (or cell or genome) may be homozygous for an inactivating mutation in the Il2rg gene. As an example, an inactivated endogenous Il2rg gene may include an insertion, deletion, or one or more point mutations in the endogenous Il2rg gene, resulting in loss of expression of a functional IL2RG protein. Some inactivated endogenous Il2rg genes may include the deletion or destruction of all endogenous Il2rg genes, or may include the deletion or destruction of an endogenous Il2rg gene fragment (i.e., a portion of a gene or a portion of a gene). For example, some, most, or all of the coding sequence in the endogenous Il2rg gene may be deleted or destroyed. In one example, the 5' fragment of the Il2rg gene may be deleted or destroyed (e.g., including the start codon). As an example, an inactivated endogenous Il2rg gene can be a gene in which the start codon of the endogenous Il2rg gene has been deleted or destroyed or mutated so that the start codon no longer functions. For example, the start codon can be destroyed by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, replacing one or more nucleotides. In another example, the 3' fragment of the Il2rg gene can be deleted or destroyed (e.g., including a stop codon). In another example, an internal fragment of the Il2rg gene (i.e., a fragment from the middle of the Il2rg gene) can be deleted or destroyed. In another example, all coding sequences in the endogenous Il2rg gene are deleted or destroyed.

[0109] Humanized Sirpa locus

[0110] In certain aspects, the transgenic non-human animals provided herein also express a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter.

[0111] Signal regulatory proteins (SIRPs) constitute a family of cell surface glycoproteins that are expressed on lymphocytes, myeloid cells (including macrophages, neutrophils, granulocytes, myeloid dendritic cells and mast cells) and neurons (e.g., see Barclay and Brown, 2006, Nat Rev Immunol 6, 457-464). Reported SIRP genes include at least SIRPA, SIRP3, SIRPβ, SIRPγ and SIRP8, and can be classified by their respective ligands and the types of signal transduction they participate in. SIRPA (also known as CD172A, SHPS1, P84, MYD-1, BIT and PTPNS1) is expressed on immune cells of the myeloid system and acts as an inhibitory receptor through an immunoreceptor tyrosine-based inhibitory motif (ITIM). SIRPA expression has also been observed on neurons. Reported SIRPA ligands include, most notably CD47, but also surfactant proteins A and D. In particular, the inhibitory effect of SIRPA in macrophage phagocytosis of host cells has been studied. For example, CD47 binds to SIRPA on macrophages to trigger inhibitory signals, thereby negatively regulating phagocytosis. Alternatively, positive signaling effects mediated by SIRPA binding have been reported (Shultz et al., 1995, J Immunol 154, 180-91). SIRPA has been shown to improve cell implantation in immunodeficient mice (Strowig et al., Proc Natl Acad Sci USA 2011; 108: 13218-13223).

[0112] The polypeptide sequence of wild-type human SIRPA and the nucleic acid sequence encoding wild-type human SIRPA can be found in Genbank accession numbers NP_001035111.1 and NM_001040022.1 (isoform 1 and transcript variant 1); NP_001035112.1 and NM_001040023.2 (isoform 1 and transcript variant 2); NP_001317657.1 and NM_001330728.1 (isoform 2 and transcript variant 4); and NP_542970.1 and NM_080792.3 (isoform 1 and transcript variant 3). The SIRPA gene is conserved in at least chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, and chickens. The genomic locus encoding the wild-type human SIRPA protein can be found at chromosome 20 of the human genome; NC_000020.11 (1894167-1940592). In some embodiments, the human SIRPA protein is encoded by exons 2 to 9 at this locus. Therefore, in some embodiments, the nucleic acid sequence comprising a coding sequence for human SIRPA includes one or more of exons 2-9 of the human SIRPA gene. In some cases, the nucleic acid sequence also includes various aspects of the human SIRPA genomic locus, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence includes the entire region of the human SIRPA genomic locus. In some cases, the nucleic acid sequence includes exons 2-4 of the human SIRPA genomic locus.

[0113] Exemplary humanized Sirpa sequences are listed in Table 3. For protein sequences, the signal peptide is underlined and the transmembrane and cytoplasmic sequences are italicized. Representative mouse Sirpa cDNA, mouse Sirpa protein, human SIRPA cDNA, and human SIRPA protein sequences are described in U.S. Pat. No. 11,019,810, which is incorporated herein by reference in its entirety.

[0114] Table 3

[0115] Humanized Sirpa protein (SEQ ID NO: 1)

[0116]

[0117]

[0118] In some embodiments, the non-human animals provided herein express humanized Sirpa proteins on the surface of immune cells (e.g., bone marrow cells) of the non-human animals, which are generated by genetic modification of endogenous sites of non-human animals encoding Sirpa proteins. Suitable examples described herein include rodents, such as mice.

[0119] In some embodiments, the humanized Sirpa gene comprises genetic material from a heterologous species (e.g., human), wherein the Sirpa protein encoded by the humanized Sirpa gene comprises a coding portion of the genetic material from the heterologous species. In some embodiments, the humanized Sirpa gene of the present disclosure comprises genomic DNA of a heterologous species, which corresponds to the extracellular portion of the SIRPA protein expressed on the cytoplasmic membrane. Non-human animals, embryos, cells, and targeting constructs for making non-human animals, non-human embryos, and cells containing the humanized Sirpa gene are also provided.

[0120] In some embodiments, the endogenous non-human animal (e.g., rodent) Sirpa gene is deleted. In some embodiments, the endogenous non-human animal (e.g., rodent) Sirpa gene is altered, wherein a portion of the endogenous non-human animal (e.g., rodent) Sirpa gene is replaced by a heterologous sequence (e.g., all or part of a human SIRPA sequence). In some embodiments, all or substantially all of the endogenous non-human animal (e.g., rodent) Sirpa gene is replaced by a heterologous gene (e.g., human SIRPA gene). In some embodiments, a portion of a heterologous SIRPα gene is inserted into an endogenous non-human Sirpa gene at an endogenous non-human animal (e.g., rodent) Sirpa locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous non-human animal (e.g., rodent) Sirpa gene is modified or humanized, thereby producing a non-human animal that is heterozygous for the humanized Sirpa gene. In other embodiments, a non-human animal is provided that is homozygous for a humanized Sirpa gene. In some embodiments, all endogenous non-human animal (e.g., rodent) Sirpa genes are replaced by a portion of a heterologous gene (e.g., a portion of a human SIRPA gene) such that the transgenic non-human animal (e.g., rodent) expresses a functional fragment of a full-length human SIRPA polypeptide (e.g., an extracellular domain of a human SIRPA polypeptide).

[0121] The non-human animals of the present disclosure contain all or part of the human SIRPA gene at the endogenous non-human Sirpa locus. Therefore, such non-human animals can be described as having heterologous SIRP genes. The SIRPα gene replaced, inserted or modified at the endogenous non-human animal (e.g., rodent) Sirpa locus can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss determination. In some embodiments, the non-human animal is heterozygous with respect to the humanized Sirpa gene.

[0122] In various embodiments, a humanized Sirpa gene according to the present disclosure includes a SIRPα gene having a second, third, and fourth exon, each exon having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the second, third, and fourth exons present in the human SIRPA gene.

[0123] In various embodiments, a humanized Sirpa gene according to the present disclosure includes a SIRPα gene having a nucleotide coding sequence (e.g., cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to nucleotides 352-1114 appearing in the human SIRPA cDNA sequence.

[0124] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an extracellular portion that has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the extracellular portion of the human SIRPA protein.

[0125] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 28-362 that appear in the human SIRPA protein.

[0126] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of the humanized SIRPA protein appearing in Table 3.

[0127] Compositions and methods for making non-human animals expressing humanized Sirpa proteins are provided, including specific polymorphic forms or allelic variants (e.g., differences in a single amino acid), including compositions and methods for making non-human animals expressing such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for making non-human animals expressing such proteins from endogenous promoters and endogenous regulatory sequences are also provided. These methods include inserting all or part of the genetic material encoding the human SIRPA protein at a precise position in the genome of the non-human animal corresponding to the endogenous non-human animal (e.g., rodent) Sirpa gene, thereby producing a humanized Sirpa gene expressing a SIRPA protein that is all or partly human. In some embodiments, these methods include inserting genomic DNA corresponding to exons 2-4 of the human SIRPA gene into the endogenous non-human animal (e.g., rodent) Sirpa gene of the non-human animal, thereby producing a humanized gene encoding a Sirpa protein, the Sirpa protein comprising a human portion containing amino acids encoded by the inserted exons.

[0128] In various embodiments, the humanized Sirpa gene approach uses relatively small modifications to the endogenous gene and produces native Sirpa-mediated signal transduction in non-human animals. Therefore, in such embodiments, the Sirpa gene modification does not affect other surrounding genes or other endogenous non-human animal (e.g., rodent) Sirp genes. In addition, in various embodiments, the modification does not affect the assembly of functional receptors on the plasma membrane, and maintains normal effector function by binding and subsequent signal transduction through the cytoplasmic portion of the receptor, which is not affected by the modification.

[0129] In addition to mice having humanized Sirpa genes as described herein, other transgenic non-human animals (e.g., rodents, such as rats) comprising humanized Sirpa genes are provided herein. In some embodiments, such non-human animals comprise a humanized Sirpa gene operably linked to an endogenous Sirpa promoter. In some embodiments, such non-human animals express a humanized Sirpa protein from an endogenous locus, wherein the humanized Sirpa protein comprises amino acid residues 28-362 of a human SIRPA protein.

[0130] Humanized Sirpa polypeptides, loci encoding humanized Sirpa polypeptides, and non-human animals expressing humanized Sirpa polypeptides are described in U.S. Patent No. 11,019,810, WO 2014 / 039782, WO 2014 / 071397, and WO 2016 / 168212, each of which is herein incorporated by reference in its entirety.

[0131] Humanized CD47 locus

[0132] In one aspect, non-human animals are provided that are genetically modified to express one or more human proteins from their genome. In certain aspects, the transgenic non-human animals provided herein also express a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0133] CD47, originally named integrin-associated protein (IAP) for its role in signal transduction from integrins on immune cells, is a transmembrane protein comprising an N-terminal immunoglobulin V (IgV) domain, five transmembrane domains and a short C-terminal intracytoplasmic tail. The length of the intracytoplasmic tail varies according to the four alternative splicing isoforms identified. CD47 (or IAP) was originally described as being expressed on all tissues (isoform 2), on neurons (isoform 4), and on keratinocytes and macrophages (isoform 1; see Reinhold et al., (1995) J. Cell Sci. 108: 3419-3425). In addition to integrins, CD47 is known to interact with several other cell surface proteins, such as, for example, thrombospondin and SIRP family members. Most notably, CD47 interacts with SIRPA and results in bidirectional signaling, thereby regulating various cell-to-cell responses, such as, for example, inhibiting phagocytosis and T cell activation. In fact, the CD47-SIRPA interaction has become the focus of attention in recent years for its role in providing tumor cells with the ability to escape immune surveillance. The combination of CD47 and SIRPA usually provides protection for normal cells through anti-phagocytic signals ("don't eat me"). However, it has been found that tumors also express anti-phagocytic signals, including CD47, to escape destruction by phagocytosis. Interestingly, CD47 is known to be upregulated in several hematological cancers and contributes to tumor growth and dissemination (Chao et al., (2012) Curr Opin Immunol. 24 (2): 225-232).

[0134] The polypeptide sequence of wild-type human CD47 and the nucleic acid sequence encoding wild-type human CD47 can be found in Genbank accession numbers NP_001369235.1 and NM_001382306.1 (isoform 3 and transcript variant 3); NP_001768.1 and NM_001777.4 (isoform 1 and transcript variant 1); NP_942088.1 and NM_198793.3 (isoform 2 and transcript variant 2); and XP_005247966.1 and XM_005247909.3 (isoform X1 and transcript variant X1). The CD47 gene is conserved at least in chimpanzees, rhesus monkeys, dogs, cattle, mice, rats and chickens. The genomic locus encoding the wild-type human CD47 protein can be found in chromosome 3 of the human genome; NC_000003.12 (c108091031-108043091). In some embodiments, the human CD47 protein is encoded by exons 1 to 11 of the locus. Therefore, in some embodiments, the nucleic acid sequence comprising the human CD47 coding sequence includes one or more of exons 1-11 of the human CD47 gene. In some cases, the nucleic acid sequence also includes various aspects of the human CD47 genomic locus, for example, introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence includes the entire region of the human CD47 genomic locus. In some cases, the nucleic acid sequence includes exons 2-7 of the human CD47 genomic locus.

[0135] Exemplary humanized CD47 sequences are listed in Table 4. For humanized protein sequences, non-human (e.g., mouse) sequences are represented in regular font, human sequences are represented in bold, and signal peptides are underlined. Representative mouse CD47 cDNA, mouse CD47 protein, human CD47 cDNA, and human CD47 protein sequences are described in U.S. Patent Publication No. 2021 / 0161112A1, which is incorporated herein by reference in its entirety.

[0136] Table 4

[0137] Humanized CD47 amino acid isoform 1 (SEQ ID NO: 2)

[0138]

[0139] Humanized CD47 amino acid isoform 2 (SEQ ID NO: 3)

[0140]

[0141] Humanized CD47 amino acid isoform 3 (SEQ ID NO: 4)

[0142]

[0143] Humanized CD47 amino acid isoform 4 (SEQ ID NO: 5)

[0144]

[0145]

[0146] In some embodiments, the non-human animals provided herein express humanized CD47 proteins on the cell surface of the non-human animal, which is generated due to genetic modification of the endogenous loci of the non-human animal encoding the CD47 protein. Suitable examples described herein include rodents, such as mice.

[0147] In some embodiments, the humanized CD47 gene comprises genetic material from a heterologous species (e.g., humans), wherein the humanized CD47 gene encodes a CD47 protein, which comprises a coding portion of genetic material from the heterologous species. In some embodiments, the humanized CD47 gene of the present disclosure comprises genomic DNA of a heterologous species encoding the extracellular portion of the CD47 protein expressed on the cytoplasmic membrane. In some embodiments, the humanized CD47 gene of the present disclosure comprises genomic DNA of a heterologous species encoding the extracellular portion and transmembrane portion of the CD47 protein expressed on the cytoplasmic membrane. Non-human animals, embryos, cells, and targeting constructs for making non-human animals, non-human embryos, and cells containing the humanized CD47 gene are also provided.

[0148] In some embodiments, the endogenous CD47 gene is deleted. In some embodiments, the endogenous CD47 gene is changed, wherein a portion of the endogenous CD47 gene is replaced by a heterologous sequence (e.g., all or part of a human CD47 sequence). In some embodiments, all or substantially all endogenous CD47 genes are replaced by heterologous genes (e.g., human CD47 genes). In some embodiments, a portion of a heterologous CD47 gene is inserted into an endogenous non-human CD47 gene at an endogenous CD47 locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous CD47 gene is modified or humanized, thereby producing a non-human animal that is heterozygous for a humanized CD47 gene. In other embodiments, a non-human animal is provided that is homozygous for a humanized CD47 gene.

[0149] In some embodiments, the non-human animals of the present disclosure contain all or part of the human CD47 gene at the endogenous non-human CD47 locus. Therefore, such non-human animals can be described as having a heterologous CD47 gene. The CD47 gene replaced, inserted or modified at the endogenous non-human animal CD47 locus can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss determination. In some embodiments, the non-human animal is heterozygous for the humanized CD47 gene. In some embodiments, the non-human animal is homozygous for the humanized CD47 gene.

[0150] In various embodiments, the humanized CD47 gene according to the present disclosure includes a CD47 gene having a second, third, fourth, fifth, sixth and seventh exons, each exon having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the second, third, fourth, fifth, sixth and seventh exons appearing in the human CD47 gene.

[0151] In various embodiments, the humanized CD47 gene according to the present disclosure includes a CD47 gene having a first exon and exons downstream of exon 7 (e.g., the eighth and ninth exons of isoform 2), each exon having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the corresponding exon appearing in the mouse CD47 gene.

[0152] In various embodiments, the humanized CD47 gene according to the present disclosure includes a CD47 gene having a 5′ untranslated region and a 3′ untranslated region, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the 5′ untranslated region and the 3′ untranslated region that appear in the mouse CD47 gene.

[0153] In various embodiments, a humanized CD47 gene according to the present disclosure includes a CD47 gene having a nucleotide coding sequence (e.g., a cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide coding sequence occurring in the human CD47 nucleotide coding sequence.

[0154] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an extracellular portion that has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the extracellular portion of a human CD47 protein.

[0155] In various embodiments, the humanized CD47 protein produced by the non-human animals of the disclosure has an extracellular portion having an amino acid sequence identical to amino acid residues 19-141 that appear in the human CD47 protein.

[0156] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain that has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the N-terminal immunoglobulin V domain of a human CD47 protein.

[0157] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain having an amino acid sequence identical to amino acid residues 19-127 that appear in the human CD47 protein.

[0158] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain and five transmembrane domains, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the N-terminal immunoglobulin V domain and five transmembrane domains of a human CD47 protein.

[0159] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an intracytoplasmic tail that has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the intracytoplasmic tail of the mouse CD47 protein.

[0160] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 16-292 that appear in human CD47 protein.

[0161] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 19-292 that appear in human CD47 protein.

[0162] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence identical to amino acid residues 19-292 (or 16-292) that appear in the human CD47 protein.

[0163] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of the humanized CD47 protein appearing in Table 4.

[0164] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is identical to the amino acid sequence of the humanized CD47 protein presented in Table 4.

[0165] Compositions and methods for making non-human animals expressing humanized CD47 proteins are provided, including specific polymorphic forms, allelic variants (e.g., single amino acid differences) or alternatively spliced ​​isoforms, including compositions and methods for making non-human animals expressing such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for making non-human animals expressing such proteins from endogenous promoters and endogenous regulatory sequences are also provided. These methods include inserting all or part of the genetic material encoding the human CD47 protein at a precise position corresponding to the endogenous CD47 gene in the genome of the non-human animal, thereby producing a humanized CD47 gene expressing all or part of the CD47 protein that is human. In some embodiments, these methods include inserting genomic DNA corresponding to exons 2-7 of the human CD47 gene into the endogenous CD47 gene of the non-human animal, thereby producing a humanized gene encoding a CD47 protein, the CD47 protein comprising a human portion containing amino acids encoded by the inserted exons.

[0166] Where appropriate, the coding region of the genetic material or polynucleotide sequence encoding the human CD47 protein may be modified in whole or in part to include codons optimized for expression in non-human animals (e.g., see U.S. Pat. Nos. 5,670,356 and 5,874,304). A codon-optimized sequence is a synthetic sequence that preferably encodes the same polypeptide encoded by a non-codon-optimized parent polynucleotide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide). In some embodiments, the coding region of the genetic material encoding the human CD47 protein may include altered sequences in whole or in part to optimize codon usage for a specific cell type (e.g., rodent cells). For example, the codons of the genomic DNA corresponding to exons 2-7 of the endogenous CD47 gene to be inserted into a non-human animal (e.g., a rodent) may be optimized for expression in non-human animal cells. Such a sequence may be described as a codon-optimized sequence.

[0167] In various embodiments, the humanized CD47 gene approach uses relatively minimal endogenous gene modification and results in natural CD47-mediated signal transduction in non-human animals because the genomic sequence of the CD47 sequence is modified in a single fragment, thereby maintaining normal functionality by including necessary regulatory sequences. Therefore, in such embodiments, the CD47 gene modification does not affect other surrounding genes or other endogenous CD47 interaction genes (e.g., thrombospondins, SIRPs, integrins, etc.). In addition, in various embodiments, the modification does not affect the assembly of the functional CD47 transmembrane protein on the plasma membrane, and maintains normal effector function by binding and subsequent signal transduction through the cytoplasmic portion of the protein, which is not affected by the modification.

[0168] Although embodiments of using humanized CD47 genes in mice are widely discussed herein (i.e., mice having a CD47 gene encoding a CD47 protein, the CD47 gene comprising a human portion and a mouse portion), other non-human animals (e.g., rodents, such as rats) comprising humanized CD47 genes are also provided. In some embodiments, such non-human animals comprise a humanized CD47 gene operably linked to an endogenous CD47 promoter. In some embodiments, such non-human animals express a humanized CD47 protein from an endogenous locus, wherein the humanized CD47 protein comprises amino acid residues 16-292 (or 19-141 or 19-127) of a human CD47 protein.

[0169] Humanized CD47 polypeptides, loci encoding humanized CD47 polypeptides, and non-human animals expressing humanized CD47 polypeptides are described in U.S. Patent Publication No. 2021 / 0161112, which is incorporated herein by reference.

[0170] Humanized M-CSF locus

[0171] In certain aspects, the transgenic non-human animals provided herein also express a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter. A human M-CSF protein refers to a protein that is human M-CSF or substantially identical to human M-CSF, for example, it is 80% or more identical to human M-CSF, 85% or more identical to human M-CSF, 90% or more identical to human M-CSF, or 95% or more identical to human M-CSF, for example, 97%, 98% or 99% identical to human M-CSF. Thus, a nucleic acid sequence encoding a human M-CSF protein is a polynucleotide comprising a coding sequence for a human M-CSF protein (i.e., human M-CSF or a protein substantially identical to human M-CSF).

[0172] M-CSF (also known as CSF-1, for "colony stimulating factor 1") is a cytokine that controls the production, differentiation and function of macrophages. The polypeptide sequence of human M-CSF and the nucleic acid sequence encoding human M-CSF can be found in Genbank accession numbers NP_000748.4 and NM_000757.6 (isoform a and transcript variant 1); NP_757349.2 and NM_172210.3 (isoform b and transcript variant 2); NP_757350.2 and NM_172211.4 (isoform c and transcript variant 3) and NP_757351.2; and NM 172212.3 (isoform a and transcript variant 4). The genomic locus encoding the human M-CSF protein can be found in chromosome 1 of the human genome; NC_000001.11 (109910506-109930992). The protein sequence is encoded by exons 1 to 8 of the locus, while exon 9 contains non-translated sequences. Therefore, a nucleic acid sequence comprising a human M-CSF coding sequence comprises one or more of exons 1-8 of the human M-CSF gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic locus of human M-CSF, such as introns, 3' and / or 5' non-translated sequences (UTRs). In some cases, the nucleic acid sequence comprises the entire region of the human M-CSF genomic locus. In some cases, the nucleic acid sequence includes exon 2 of the human M-CSF genomic locus to 633 nucleotides downstream of non-coding exon 9.

[0173] In some embodiments, in the transgenic non-human animals provided herein, the nucleic acid sequence encoding the human M-CSF protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) M-CSF gene. The non-human animal (e.g., mouse) M-CSF regulatory sequences are those sequences of the non-human animal (e.g., mouse) M-CSF genomic site that regulate the expression of non-human animal (e.g., mouse) M-CSF, such as 5' regulatory sequences, e.g., M-CSF promoter, M-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., 3' UTR; and enhancers, etc. For example, mouse M-CSF is located on chromosome 3, NC_000069.7, at approximately position c107668048-107648364, and the mouse M-CSF coding sequence can be found in Genbank accession numbers NM_007778.4 (encoding transcript variant 1 of isoform 1), NM_001113529.1 (encoding transcript variant 2 of isoform 2), and NM 001113530.1 (encoding transcript variant 3 of isoform 1). Regulatory sequences for mouse M-CSF are well defined in the art and can be readily identified using computer methods, for example, by reference to the above Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, or by experimental methods described in the art, for example, Abboud et al., (2003) Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model. J. Histochemistry and Cytochemistry 51(7):941-949, the disclosure of which is incorporated herein by reference. In some cases, for example, when the nucleic acid sequence encoding the human M-CSF protein is located at the non-human animal (e.g., mouse) M-CSF genomic locus, the regulatory sequences operably linked to the human CSF coding sequence are sequences endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they are present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0174] In some examples, transgenic non-human animals expressing human M-CSF protein are generated by random integration or insertion of a human nucleic acid sequence encoding a human M-CSF protein or a fragment thereof, i.e., a "human M-CSF nucleic acid sequence" or "human M-CSF sequence", into the genome of a non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human M-CSF protein in the genome is unknown. In other cases, transgenic non-human animals expressing human M-CSF protein are generated by targeted integration or insertion of a human M-CSF nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at a target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Thus, for example, in a transgenic non-human animal (e.g., mouse) comprising a nucleic acid sequence encoding a human M-CSF protein created by targeting a human M-CSF nucleic acid sequence to a non-human animal M-CSF (e.g., mouse) locus, the human M-CSF nucleic acid sequence can replace part or all of the non-human animal (e.g., mouse) sequence at the M-CSF locus, e.g., exons and / or introns. In some such examples, the human M-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) M-CSF locus such that expression of the human M-CSF sequence is regulated by a native or endogenous regulatory sequence at the non-human animal (e.g., mouse) M-CSF locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human M-CSF protein is operably linked is a native M-CSF regulatory sequence of the non-human animal (e.g., mouse) M-CSF locus.

[0175] In some cases, the integration of the human M-CSF sequence will not affect the transcription of the gene into which the human M-CSF sequence has been integrated. For example, if the human M-CSF sequence is integrated into the coding sequence as an intein, or the human M-CSF sequence contains a 2A peptide, the human M-CSF sequence will be transcribed and translated simultaneously with the gene into which the human M-CSF sequence has been integrated. In other cases, the integration of the human M-CSF sequence will interrupt the transcription of the gene into which the human M-CSF sequence has been integrated. For example, when the human M-CSF sequence is integrated by homologous recombination, part or all of the coding sequence at the integration site may be removed, thereby transcribing the human M-CSF sequence. In some such examples, the integration of the human M-CSF sequence will produce a null mutation, and thus a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of the gene. This can be the result of the complete absence of a gene product (protein, RNA) or the expression of a non-functional gene product at the molecular level. At the phenotypic level, a null allele is indistinguishable from the deletion of the entire locus.

[0176] In some examples, a transgenic non-human animal (e.g., mouse) expressing a human M-CSF protein comprises a copy of a nucleic acid sequence encoding a human M-CSF protein. For example, a non-human animal (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at one locus will comprise the nucleic acid sequence, and the other allele will be an endogenous allele. For example, as described above, in some cases, a human M-CSF nucleic acid sequence is integrated into a non-human animal (e.g., mouse) M-CSF locus, thereby generating a null allele of non-human animal (e.g., mouse) M-CSF. In some such embodiments, a humanized M-CSF mouse may be heterozygous for a nucleic acid coding sequence, i.e., a humanized M-CSF mouse comprises a null allele of non-human animal (e.g., mouse) M-CSF (an allele comprising the nucleic acid sequence) and an endogenous M-CSF allele (wild type or other). In other cases, the transgenic non-human animal (e.g., mouse) expressing the human M-CSF protein comprises two copies of the nucleic acid sequence encoding the human M-CSF protein. For example, the non-human animal (e.g., mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles of one locus in the diploid genome will comprise the nucleic acid sequence, i.e., the transgenic non-human animal (e.g., mouse) expressing the human M-CSF protein comprises two null alleles of mouse M-CSF (alleles comprising the nucleic acid sequence).

[0177] Although embodiments using a human M-CSF gene in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising a human M-CSF gene are also provided.

[0178] WO 2012 / 112544, WO 2014 / 039782, and WO 2014 / 071397 describe human M-CSF polypeptides, loci encoding human M-CSF polypeptides, and non-human animals expressing human M-CSF polypeptides, each of which is incorporated herein by reference.

[0179] Humanized GM-CSF locus

[0180] In some aspects, the transgenic non-human animals provided herein also express a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter. A human GM-CSF protein refers to a protein that is human GM-CSF or substantially identical to human GM-CSF, for example, it is 80% or more identical to human GM-CSF, 85% or more identical to human GM-CSF, 90% or more identical to human GM-CSF, or 95% or more identical to human GM-CSF, for example, 97%, 98% or 99% identical to human GM-CSF. Thus, a nucleic acid sequence encoding a human GM-CSF protein is a polynucleotide comprising a coding sequence for a human GM-CSF protein (i.e., human GM-CSF or a protein substantially identical to human GM-CSF).

[0181] GM-CSF is a cytokine that is essential for the development and function of myeloid cells. GM-CSF has no cross-reactivity between humans and mice. GM-CSF is highly expressed in the lungs and is important for lung homeostasis in vivo, as evidenced by the fact that GM-CSF KO mice develop pulmonary alveolar proteinosis (PAP), which is characterized by the accumulation of proteins in the lungs due to surfactant clearance defects. Alveolar macrophages from GM-CSF KO mice have terminal differentiation defects, which leads to impaired innate immunity to pulmonary pathogens. GM-CSF also stimulates the proliferation of human alveolar macrophages (AM) in vitro. GM-CSF is largely non-essential for homeostatic hematopoiesis. In contrast, GM-CSF is required for inflammatory responses, such as the production of proinflammatory cytokines by macrophages and the mobilization and recruitment of monocytes. GM-CSF is also essential for protective immunity against a range of pathogens, including Mycobacterium tuberculosis. In particular, GM-CSF KO mice infected with Mycobacterium tuberculosis do not develop granulomas, which are the hallmarks of tuberculosis.

[0182] The polypeptide sequence of human GM-CSF and the nucleic acid sequence encoding human GM-CSF can be found in Genbank accession numbers NP_000749.2 and NM_000758.4, respectively. The genomic locus encoding the human GM-CSF protein can be found in chromosome 5 of the human genome; NG_033024.1 (4998-7379). The protein sequence is encoded by exons 1 to 4 at this locus. Therefore, a nucleic acid sequence comprising a human GM-CSF coding sequence comprises one or more of exons 1-4 of the human GM-CSF gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic locus of human GM-CSF, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence comprises the entire region of the human GM-CSF genomic locus.

[0183] In some embodiments, in the transgenic non-human animals provided herein, the nucleic acid sequence encoding the human GM-CSF protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) GM-CSF gene. The non-human animal (e.g., mouse) GM-CSF regulatory sequences are those sequences of the non-human animal (e.g., mouse) GM-CSF genomic loci that regulate the expression of non-human animal (e.g., mouse) GM-CSF, such as 5' regulatory sequences, e.g., GM-CSF promoter, GM-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., 3' UTR; and enhancers, etc. For example, mouse GM-CSF is located on chromosome 11, GRCm39, NC_000077.7, approximately at position c54140725-54138096, and the mouse GM-CSF coding sequence can be found in Genbank accession number NM_009969.4. Regulatory sequences for mouse M-CSF are well defined in the art and can be readily identified using computer methods, for example, by reference to the above-mentioned Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human GM-CSF protein is located at the non-human animal (e.g., mouse) GM-CSF genomic locus, the regulatory sequences operably linked to the human GM-CSF coding sequence are sequences endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they are present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0184] In some examples, transgenic non-human animals expressing human GM-CSF protein are generated by random integration or insertion of a human nucleic acid sequence encoding a human GM-CSF protein or a fragment thereof, i.e., a "human GM-CSF nucleic acid sequence" or "human GM-CSF sequence", into the genome of a non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human GM-CSF protein in the genome is unknown. In other cases, transgenic non-human animals expressing human GM-CSF protein are generated by targeted integration or insertion of a human GM-CSF nucleic acid sequence into the genome of a non-human animal, such as by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at a target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Thus, for example, in a transgenic non-human animal (e.g., mouse) comprising a nucleic acid sequence encoding a human GM-CSF protein created by targeting a human GM-CSF nucleic acid sequence to a non-human animal GM-CSF (e.g., mouse) locus, the human GM-CSF nucleic acid sequence can replace part or all of the non-human animal (e.g., mouse) sequence at the GM-CSF locus, e.g., exons and / or introns. In some such examples, the human GM-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) GM-CSF locus such that expression of the human GM-CSF sequence is regulated by a native or endogenous regulatory sequence at the non-human animal (e.g., mouse) GM-CSF locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human GM-CSF protein is operably linked is a native GM-CSF regulatory sequence of the non-human animal (e.g., mouse) GM-CSF locus.

[0185] In some cases, the integration of the human GM-CSF sequence will not affect the transcription of the gene into which the human GM-CSF sequence has been integrated. For example, if the human GM-CSF sequence is integrated into the coding sequence as an intein, or the human GM-CSF sequence contains a 2A peptide, the human GM-CSF sequence will be transcribed and translated simultaneously with the gene into which the human GM-CSF sequence has been integrated. In other cases, the integration of the human GM-CSF sequence will interrupt the transcription of the gene into which the human GM-CSF sequence has been integrated. For example, when the human GM-CSF sequence is integrated by homologous recombination, part or all of the coding sequence at the integration site may be removed, thereby transcribing the human GM-CSF sequence. In some such examples, the integration of the human GM-CSF sequence will produce a null mutation, and thus a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of the gene. This can be the result of the complete absence of a gene product (protein, RNA) or the expression of a non-functional gene product at the molecular level. At the phenotypic level, a null allele is indistinguishable from the deletion of the entire locus.

[0186] In some examples, a transgenic non-human animal (e.g., mouse) expressing a human GM-CSF protein comprises a copy of a nucleic acid sequence encoding a human GM-CSF protein. For example, a non-human animal (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at one locus will comprise the nucleic acid sequence, while the other allele will be an endogenous allele. For example, as described above, in some cases, a human GM-CSF nucleic acid sequence is integrated into a non-human animal (e.g., mouse) GM-CSF locus, thereby generating a null allele of GM-CSF in a non-human animal (e.g., mouse). In some such embodiments, a humanized GM-CSF mouse may be heterozygous for a nucleic acid coding sequence, i.e., a humanized GM-CSF mouse comprises a null allele of GM-CSF in a non-human animal (e.g., mouse) (an allele comprising the nucleic acid sequence) and an endogenous GM-CSF allele (wild type or other). In other cases, the transgenic non-human animal (e.g., mouse) expressing the human GM-CSF protein comprises two copies of the nucleic acid sequence encoding the human GM-CSF protein. For example, the non-human animal (e.g., mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles of one locus in the diploid genome will comprise the nucleic acid sequence, i.e., the transgenic non-human animal (e.g., mouse) expressing the human GM-CSF protein comprises two null alleles of mouse GM-CSF (alleles comprising the nucleic acid sequence).

[0187] Although embodiments using the human GM-CSF gene in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising the human GM-CSF gene are also provided.

[0188] WO 2011 / 044050, WO 2014 / 039782, and WO 2014 / 071397 describe human GM-CSF polypeptides, loci encoding human GM-CSF polypeptides, and non-human animals expressing human GM-CSF polypeptides, each of which is incorporated herein by reference.

[0189] Humanized TPO locus

[0190] In certain aspects, the transgenic non-human animals provided herein also express a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter. A human TPO protein refers to a protein that is human TPO or substantially identical to human TPO, for example, it is 80% or more identical to human TPO, 85% or more identical to human TPO, 90% or more identical to human TPO, or 95% or more identical to human TPO, for example, 97%, 98% or 99% identical to human TPO. Therefore, a nucleic acid sequence encoding a human TPO protein is a polynucleotide comprising a coding sequence for a human TPO protein (i.e., human TPO or a protein substantially identical to human TPO).

[0191] Thrombopoietin (TPO) was originally identified as a growth factor that promotes the development of megakaryocytes and platelets. TPO is constitutively produced by the liver and kidneys and released into the blood circulation. The TPO receptor c-Mpl is expressed by hematopoietic stem and progenitor cells in the bone marrow. C-Mpl is also expressed on circulating platelets. However, the binding of TPO on platelets does not activate any signaling pathways. Therefore, platelets act as a sink or scavenger for TPO and promote negative regulation of platelet production through this mechanism. Subsequently, TPO has been recognized for its important function in supporting HSC expansion and self-renewal. TPO deficiency leads to a reduction in the number of HSCs in adult mice, and the presence of TPO is required to maintain adult HSCs in a quiescent state. In addition, TPO is required to support the post-transplantation expansion of HSCs, which is required to replenish the hematopoietic compartment of irradiated hosts. Interestingly, it has been shown that osteoblasts involved in the formation of HSC niches in the bone marrow produce TPO, which is essential for the function and maintenance of HSCs.

[0192] The polypeptide sequence of human TPO and the nucleic acid sequence encoding human TPO can be found in Genbank accession numbers NM_000547.6 and NP_000538.3 (transcript variant 1 and isoform a); NM_001206744.2 and NP_001193673.1 (transcript variant 6 and isoform a); NM_001206745.2 and NP_001193674.1 (transcript variant 7 and isoform b); NM_175719.4 and NP_783650.1 (transcript variant 2 and isoform b); NM_175721.3 and NP_783652.1 (transcript variant 4 and isoform d); and NM_175722.3 and NP_783653.1 (transcript variant 5 and isoform e). The genomic locus encoding the human TPO protein can be found in chromosome 2 of the human genome; NG_011581.1 (4999-134265). The protein sequence is encoded by exons 2 to 17 of this locus. Therefore, the nucleic acid sequence comprising the human TPO coding sequence comprises one or more of exons 2-17 of the human TPO gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic locus of human TPO, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence comprises the entire region of the human TPO genomic locus.

[0193] In some embodiments, in the transgenic non-human animals provided herein, the nucleic acid sequence encoding human TPO protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) TPO gene. Non-human animal (e.g., mouse) TPO regulatory sequences are those sequences of the non-human animal (e.g., mouse) TPO genomic site that regulates the expression of non-human animal (e.g., mouse) TPO, such as 5' regulatory sequences, e.g., TPO promoter, TPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., 3' UTR; and enhancers, etc. For example, mouse TPO is located at chromosome 12, GRCm39, NC_000078.7, approximately at the position of c30182983-30104658, and the mouse TPO coding sequence can be found in Genbank accession number NM_009417.3. The regulatory sequences of mouse TPO are well defined in the art and can be readily identified using computer methods, for example, by reference to the above-mentioned Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human TPO protein is located at the non-human animal (e.g., mouse) TPO genomic locus, the regulatory sequences operably linked to the human TPO coding sequence are endogenous or native sequences of the non-human animal (e.g., mouse) genome, that is, they are present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0194] In some examples, transgenic non-human animals expressing human TPO protein are produced by randomly integrating or inserting a human nucleic acid sequence encoding a human TPO protein or a fragment thereof, i.e., a "human TPO nucleic acid sequence" or a "human TPO sequence", into the genome of a non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human TPO protein in the genome is unknown. In other cases, transgenic non-human animals expressing human TPO protein are produced by targeted integration or insertion of a human TPO nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Thus, for example, in a transgenic non-human animal (e.g., mouse) comprising a nucleic acid sequence encoding a human TPO protein created by targeting a human TPO nucleic acid sequence to a non-human animal TPO (e.g., mouse) locus, the human TPO nucleic acid sequence can replace part or all of the non-human animal (e.g., mouse) sequence at the TPO locus, e.g., exons and / or introns. In some such examples, the human TPO nucleic acid sequence is integrated into the non-human animal (e.g., mouse) TPO locus so that the expression of the human TPO sequence is regulated by a natural or endogenous regulatory sequence at the non-human animal (e.g., mouse) TPO locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human TPO protein is operably linked is a natural TPO regulatory sequence at the non-human animal (e.g., mouse) TPO locus.

[0195] In some cases, the integration of the human TPO sequence will not affect the transcription of the gene into which the human TPO sequence has been integrated. For example, if the human TPO sequence is integrated into the coding sequence as an intein, or the human TPO sequence contains a 2A peptide, the human TPO sequence will be transcribed and translated simultaneously with the gene into which the human TPO sequence has been integrated. In other cases, the integration of the human TPO sequence interrupts the transcription of the gene into which the human TPO sequence has been integrated. For example, when the human TPO sequence is integrated by homologous recombination, part or all of the coding sequence at the integration site can be removed, thereby transcribing the human TPO sequence. In some such instances, the integration of the human TPO sequence will produce invalid mutations, and therefore invalid alleles will be produced. Invalid alleles are mutant copies of genes that completely lack the normal function of the gene. This can be the result of the complete absence of gene products (proteins, RNA) or the expression of non-functional gene products at the molecular level. At the phenotypic level, invalid alleles are indistinguishable from the absence of the entire locus.

[0196] In some instances, a transgenic non-human animal (e.g., mouse) expressing human TPO protein comprises a copy of a nucleic acid sequence encoding human TPO protein. For example, a non-human animal (e.g., mouse) can be heterozygous for a nucleic acid sequence. In other words, an allele on a locus will comprise the nucleic acid sequence, and another allele will be an endogenous allele. For example, as described above, in some cases, a human TPO nucleic acid sequence is integrated into a non-human animal (e.g., mouse) TPO locus, thereby producing an invalid allele of non-human animal (e.g., mouse) TPO. In some such embodiments, a humanized TPO mouse can be heterozygous for a nucleic acid coding sequence, that is, a humanized TPO mouse comprises an invalid allele (an allele comprising the nucleic acid sequence) of a non-human animal (e.g., mouse) TPO and an endogenous TPO allele (wild type or other). In other cases, a transgenic non-human animal (e.g., mouse) expressing human TPO protein comprises two copies of a nucleic acid sequence encoding human TPO protein. For example, a non-human animal (e.g., a mouse) can be homozygous for a nucleic acid sequence, i.e., both alleles of one locus in the diploid genome will comprise the nucleic acid sequence, i.e., a transgenic non-human animal (e.g., a mouse) expressing human TPO protein comprises two null alleles of mouse TPO (alleles comprising the nucleic acid sequence).

[0197] Although embodiments using a human TPO gene in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising a human TPO gene are also provided.

[0198] WO 2011 / 044050, WO 2014 / 039782, and WO 2014 / 071397 describe human TPO polypeptides, loci encoding human TPO polypeptides, and non-human animals expressing human TPO polypeptides, each of which is incorporated herein by reference.

[0199] Humanized EPO locus

[0200] In some aspects, transgenic non-human animals provided herein also express human erythropoietin (hEPO) protein encoded by a nucleic acid operably connected to an EPO promoter. Human EPO protein refers to human EPO or a protein substantially identical to human EPO, for example, it is 80% or more identical to human EPO, 85% or more identical, 90% or more identical or 95% or more identical, for example, 97%, 98% or 99% identical to human EPO. Therefore, the nucleic acid sequence encoding human EPO protein is a polynucleotide comprising the coding sequence of human EPO protein (that is, human EPO or a protein substantially identical to human EPO).

[0201] Erythropoietin (EPO) encodes a secreted glycosylated cytokine composed of four alpha-helical bundles. The encoded EPO protein is primarily synthesized in the kidney, secreted into the plasma, and binds to the erythropoietin receptor to promote the production of red blood cells or erythropoiesis in the bone marrow. Under hypoxic conditions, expression of the EPO gene is upregulated, resulting in increased erythropoiesis and enhanced oxygen-carrying capacity of the blood. Expression of the EPO gene has also been observed in the brain and eyes, and elevated expression levels have been observed in diabetic retinopathy and ocular hypertension. Recombinant forms of the encoded EPO protein exhibit neuroprotective activity against a variety of potential brain insults, as well as anti-apoptotic functions in multiple tissue types, and have been used to treat anemia and enhance the efficacy of cancer therapy.

[0202] The polypeptide sequence of human EPO and the nucleic acid sequence encoding human EPO can be found in Genbank accession numbers NP_000790.2 and NM_000799.4, respectively. The genomic site encoding human EPO protein can be found in chromosome 7 of the human genome, NG_021471.2 (4669-7901) or NC_000007.14 (100720468-100723700). The protein sequence is encoded by exons 1 to 5 at the site. Therefore, the nucleic acid sequence comprising the human EPO coding sequence comprises one or more of exons 1-5 of the human EPO gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic site of human EPO, such as introns, 3' and / or 5' untranslated sequences (UTR). In some cases, the nucleic acid sequence comprises the entire region of the human EPO genomic site.

[0203] In some embodiments, in the transgenic non-human animals provided herein, the nucleic acid sequence encoding human EPO protein is operably connected to one or more regulatory sequences of non-human animal (e.g., mouse) EPO gene. Non-human animal (e.g., mouse) EPO regulatory sequences are those sequences of non-human animal (e.g., mouse) EPO genomic sites that regulate non-human animal (e.g., mouse) EPO expression, such as 5' regulatory sequences, for example, EPO promoter, EPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, for example, 3' UTR; and enhancers, etc. For example, mouse EPO is located at chromosome 5, GRCm39, NC_000071.7, approximately at the position of c137484078-137481282, and mouse EPO encoding sequences can be found in Genbank accession numbers NM_007942.2 (transcript variant 1 encoding isoform 1) and NM_001312875.1 (transcript variant 2 encoding isoform 2). The regulatory sequences of mouse EPO are well defined in the art and can be easily identified using computer methods, for example, by reference to the above-mentioned Genbank accession number on the UCSC genome browser on the World Wide Web genome.ucsc.edu, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding human EPO protein is located at the non-human animal (e.g., mouse) EPO genome site, the regulatory sequence operably connected to the human EPO coding sequence is an endogenous or natural sequence of the non-human animal (e.g., mouse) genome, that is, they are present in the non-human animal (e.g., mouse) genome before integrating the human nucleic acid sequence.

[0204] In some examples, transgenic non-human animals expressing human EPO protein are randomly integrated or inserted into the genome of non-human animals by encoding human nucleic acid sequences or fragments thereof, i.e., "human EPO nucleic acid sequences" or "human EPO sequences". Typically, in such embodiments, the position of the nucleic acid sequence encoding human EPO protein in the genome is unknown. In other cases, transgenic non-human animals expressing human EPO protein are produced by targeted integration or insertion of human EPO nucleic acid sequences into the genome of non-human animals, such as by homologous recombination. In homologous recombination, polynucleotides are inserted into the host genome at the target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Therefore, for example, in a transgenic non-human animal (for example, mouse) comprising a nucleic acid sequence encoding human EPO protein created by targeting human EPO nucleic acid sequence to non-human animal EPO (for example, mouse) locus, human EPO nucleic acid sequence can replace part or all of non-human animal (for example, mouse) sequence at EPO locus, for example, exon and / or intron. In some such examples, human EPO nucleic acid sequence is integrated into non-human animal (for example, mouse) EPO locus so that the expression of human EPO sequence is regulated by natural or endogenous regulatory sequence at non-human animal (for example, mouse) EPO locus. In other words, the regulatory sequence operably connected to the nucleic acid sequence encoding human EPO protein is the natural EPO regulatory sequence at non-human animal (for example, mouse) EPO locus.

[0205] In some cases, the integration of human EPO sequence will not affect the transcription of the gene in which the human EPO sequence has been integrated. For example, if the human EPO sequence is integrated into the coding sequence as an intein, or the human EPO sequence includes a 2A peptide, the human EPO sequence will be transcribed and translated simultaneously with the gene in which the human EPO sequence has been integrated. In other cases, the integration of the human EPO sequence interrupts the transcription of the gene in which the human EPO sequence has been integrated. For example, when the human EPO sequence is integrated by homologous recombination, some or all of the coding sequence at the integration site can be removed, so as to transcribe the human EPO sequence. In some such examples, the integration of the human EPO sequence will produce null mutations, so null alleles will be produced. Null alleles are mutant copies of genes that lack the normal function of the gene completely. This can be due to the complete absence of gene products (proteins, RNA) or the expression of non-functional gene products at the molecular level. At the phenotypic level, null alleles are indistinguishable from the absence of the entire locus.

[0206] In some instances, the transgenic non-human animal (e.g., mouse) expressing human EPO protein includes a copy of the nucleotide sequence encoding human EPO protein. For example, non-human animals (e.g., mice) can be heterozygous for nucleotide sequence. In other words, an allele on a locus will include the nucleotide sequence, and another allele will be an endogenous allele. For example, as described above, in some cases, the human EPO nucleotide sequence is integrated into the non-human animal (e.g., mouse) EPO locus, thereby producing the invalid allele of non-human animal (e.g., mouse) EPO. In some such embodiments, humanized EPO mice can be heterozygous for nucleic acid coding sequences, that is, humanized EPO mice include an invalid allele (including the allele of the nucleotide sequence) and an endogenous EPO allele (wild type or other) of a non-human animal (e.g., mouse) EPO. In other cases, the transgenic non-human animal (e.g., mouse) expressing human EPO protein includes two copies of the nucleotide sequence encoding human EPO protein. For example, a non-human animal (e.g., a mouse) can be homozygous for a nucleic acid sequence, i.e., both alleles of a locus in a diploid genome will comprise the nucleic acid sequence, i.e., a transgenic non-human animal (e.g., a mouse) expressing human EPO protein comprises two null alleles of mouse EPO (alleles comprising the nucleic acid sequence).

[0207] Although embodiments using human EPO genes in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising human EPO genes are also provided.

[0208] WO 2015 / 179317 describes human EPO polypeptides, loci encoding human EPO polypeptides, and non-human animals expressing human EPO polypeptides, and the application is incorporated herein by reference.

[0209] Humanized IL-3 locus

[0210] In certain aspects, the transgenic non-human animals provided herein also express human IL-3 proteins encoded by nucleic acids operably linked to IL-3 promoters. Human IL-3 proteins refer to proteins that are human IL-3 or substantially identical to human IL-3, for example, they are 80% or more identical to human IL-3, 85% or more identical, 90% or more identical or 95% or more identical, for example, 97%, 98% or 99% identical to human IL-3. Therefore, the nucleic acid sequence encoding human IL-3 proteins is a polynucleotide comprising the coding sequence of human IL-3 proteins (i.e., human IL-3 or proteins substantially identical to human IL-3).

[0211] Like GM-CSF, IL-3 is a cytokine that is essential for the development and function of myeloid cells. IL-3 does not cross-react between humans and mice. IL-3 stimulates early hematopoietic progenitor cells in vitro but is not essential for homeostatic hematopoiesis in vivo. However, together with GM-CSF, it is required for an effective DTH response in vivo. IL-3 also specifically stimulates the proliferation of alveolar macrophages (AM) in vitro.

[0212] The polypeptide sequence of human IL-3 and the nucleic acid sequence encoding human IL-3 can be found in Genbank accession numbers NP_000579.2 and NM_000588.4. The genomic site encoding the human IL-3 protein can be found in chromosome 5 of the human genome, GRCh38.p14; NC_000005.10 (132060655-132063204). The protein sequence is encoded by exons 1 to 5 at this site. Therefore, the nucleic acid sequence comprising the human IL-3 coding sequence comprises one or more of exons 1-5 of the human IL-3 gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic site of human IL-3, such as introns, 3' and / or 5' untranslated sequences (UTR). In some cases, the nucleic acid sequence comprises the entire region of the human IL-3 genomic site.

[0213] In some embodiments, in the transgenic non-human animals provided herein, the nucleic acid sequence encoding the human IL-3 protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) IL-3 gene. Non-human animal (e.g., mouse) IL-3 regulatory sequences are those sequences of the non-human animal (e.g., mouse) IL-3 genomic sites that regulate the expression of non-human animal (e.g., mouse) IL-3, such as 5' regulatory sequences, e.g., IL-3 promoter, IL-3 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., 3' UTR; and enhancers, etc. For example, mouse IL-3 is located at chromosome 11, GRCm39, NC_000077.7, approximately at the position of c54158105-54155911, and the mouse IL-3 coding sequence can be found in Genbank accession number NM_010556.4. The regulatory sequences of mouse IL-3 are well defined in the art and can be readily identified using computer methods, for example, by reference to the above-mentioned Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human IL-3 protein is located at the non-human animal (e.g., mouse) IL-3 genomic locus, the regulatory sequences operably linked to the human IL-3 coding sequence are sequences endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they are present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0214] In some examples, transgenic non-human animals expressing human IL-3 proteins are produced by random integration or insertion of a human nucleic acid sequence encoding a human IL-3 protein or a fragment thereof, i.e., a "human IL-3 nucleic acid sequence" or a "human IL-3 sequence", into the genome of a non-human animal. Typically, in such embodiments, the position of the nucleic acid sequence encoding the human IL-3 protein in the genome is unknown. In other cases, transgenic non-human animals expressing human IL-3 proteins are produced by targeted integration or insertion of a human IL-3 nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Therefore, for example, in a transgenic non-human animal (e.g., mouse) comprising a nucleic acid sequence encoding a human IL-3 protein created by targeting a human IL-3 nucleic acid sequence to a non-human animal IL-3 (e.g., mouse) locus, a human IL-3 nucleic acid sequence can replace part or all of a non-human animal (e.g., mouse) sequence at the IL-3 locus, e.g., exons and / or introns. In some such examples, a human IL-3 nucleic acid sequence is integrated into a non-human animal (e.g., mouse) IL-3 locus so that the expression of the human IL-3 sequence is regulated by a natural or endogenous regulatory sequence at the non-human animal (e.g., mouse) IL-3 locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human IL-3 protein is operably connected is a natural IL-3 regulatory sequence at a non-human animal (e.g., mouse) IL-3 locus.

[0215] In some cases, the integration of human IL-3 sequences will not affect the transcription of genes into which human IL-3 sequences have been integrated. For example, if the human IL-3 sequence is integrated into the coding sequence as an intein, or the human IL-3 sequence comprises a 2A peptide, the human IL-3 sequence will be transcribed and translated simultaneously with the gene into which the human IL-3 sequence has been integrated. In other cases, the integration of human IL-3 sequences interrupts the transcription of genes into which human IL-3 sequences have been integrated. For example, when human IL-3 sequences are integrated by homologous recombination, some or all of the coding sequences at the integration site can be removed, thereby transcribing human IL-3 sequences. In some such examples, the integration of human IL-3 sequences can produce invalid mutations, and therefore invalid alleles can be produced. Invalid alleles are mutant copies of genes that completely lack the normal function of the gene. This can be due to the complete absence of gene products (proteins, RNA) or the expression of non-functional gene products at the molecular level. At the phenotypic level, invalid alleles are indistinguishable from the absence of entire loci.

[0216] In some instances, the transgenic non-human animal (e.g., mouse) expressing human IL-3 protein comprises a copy of the nucleic acid sequence encoding human IL-3 protein. For example, non-human animals (e.g., mice) can be heterozygous for nucleic acid sequences. In other words, an allele on a locus will comprise the nucleic acid sequence, and another allele will be an endogenous allele. For example, as described above, in some cases, human IL-3 nucleic acid sequences are integrated into non-human animal (e.g., mouse) IL-3 locus, thereby producing the invalid allele of non-human animal (e.g., mouse) IL-3. In some such embodiments, humanized IL-3 mice can be heterozygous for nucleic acid coding sequences, that is, humanized IL-3 mice comprise an invalid allele (comprising the allele of the nucleic acid sequence) and an endogenous IL-3 allele (wild type or other) of a non-human animal (e.g., mouse) IL-3. In other cases, transgenic non-human animals (e.g., mice) expressing human IL-3 protein comprise two copies of the nucleic acid sequence encoding human IL-3 protein. For example, a non-human animal (e.g., a mouse) can be homozygous for a nucleic acid sequence, i.e., both alleles of one locus in the diploid genome will comprise the nucleic acid sequence, i.e., a transgenic non-human animal (e.g., a mouse) expressing human IL-3 protein comprises two null alleles of mouse IL-3 (alleles comprising the nucleic acid sequence).

[0217] Although embodiments using human IL-3 genes in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising human IL-3 genes are also provided.

[0218] WO 2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397 describe human IL-3 polypeptides, loci encoding human IL-3 polypeptides and non-human animals expressing human IL-3 polypeptides, each of which is incorporated herein by reference.

[0219] Humanized IL15 locus

[0220] In certain aspects, the transgenic non-human animals provided herein also express a human IL-15 protein encoded by a nucleic acid operably linked to an IL-15 promoter. As used herein, "human IL-15 protein" refers to a protein that is a wild-type (or native) human IL-15 protein or a variant of a wild-type (or native) human IL-15 protein, which retains one or more signal transduction functions of the wild-type (or native) human IL-15 protein, for example, allowing stimulation of the human IL-15 receptor (or signal transduction through the human IL-15 receptor), and / or capable of binding to the human IL-15 receptor α subunit of the human IL-15 receptor, and / or capable of binding to IL-2Rβ / IL-15Rβ and the common γ chain (γc). The term "human IL-15 protein" also includes fragments of wild-type human IL-15 protein (or variants thereof) that retain one or more signaling functions of the wild-type human IL-15 protein, for example, fragments of human IL-15 protein that allow stimulation of the human IL-15 receptor (or signaling through the human IL-15 receptor), and / or are capable of binding to the human IL-15 receptor α subunit of the human IL-15 receptor, and / or are capable of binding to IL-2Rβ / IL-15Rβ and the common γ chain (γc).

[0221] The term "human IL-15 protein" also includes fusion proteins, i.e. chimeric proteins, which include one or more fragments of wild-type human IL-15 protein (or its variant), and they retain one or more signal transduction functions of wild-type human IL-15 protein, such as described above. Fusion proteins including one or more fragments of wild-type human IL-15 protein (or its variant) may also be referred to as humanized IL-15 proteins herein.

[0222] Therefore, the nucleic acid sequence encoding the human IL-15 protein is a polynucleotide comprising the coding sequence of the human IL-15 protein, i.e., a wild-type human IL-15 protein, a variant of the wild-type human IL-15 protein, a fragment of the wild-type human IL-15 protein (or a variant thereof) which retains one or more signal transduction functions of the wild-type human IL-15 protein, or a fusion protein, i.e., a chimeric protein, which comprises one or more fragments of the wild-type human IL-15 protein (or a variant thereof) and retains one or more signal transduction functions of the wild-type human IL-15 protein, e.g., as described above.

[0223] IL-15 (also known as "interleukin 15") is a cytokine that stimulates the proliferation of T lymphocytes. The polypeptide sequence of wild-type human IL-15 and the nucleic acid sequence encoding wild-type human IL-15 can be found in Genbank accession numbers NP_000576.1 and NM_000585.5 (isoform 1 and transcript variant 3), NP 751915.1 and NM_172175.3 (isoform 2 and transcript variant 2). The genomic locus encoding the wild-type human IL-15 protein can be found in chromosome 4 of the human genome; NC000004.12 (141636583-141733987) or NG_029605.2 (4988-102392). In some embodiments, the human IL-15 locus (e.g., NM_000585.5) includes 8 exons, of which exons 3-8 are coding exons. Therefore, in some embodiments, the nucleic acid sequence comprising the human IL-15 coding sequence comprises one or more of the exons 3-8 of the human IL-15 gene. In some cases, the nucleic acid sequence also includes various aspects of the genomic site of human IL-15, such as introns, 3' and / or 5' untranslated sequences (UTR). In some cases, the nucleic acid sequence comprises the entire region of the human IL-15 genomic site. In some cases, the nucleic acid sequence includes exons 5-8 (e.g., encoding exons 3-6) of the human IL-15 genomic site.

[0224] In the humanized IL-15 non-human animals described herein, the nucleic acid sequence encoding the human IL-15 protein is operably linked to one or more regulatory sequences of the IL-15 gene, for example, the regulatory sequence of the non-human animal IL-15 gene. Non-human animal (e.g., mouse) IL-15 regulatory sequences are those sequences of the non-human animal IL-15 genomic sites that regulate the expression of non-human animal IL-15, such as 5' regulatory sequences, for example, IL-15 promoter, IL-15 5' untranslated region (UTR), etc.; 3' regulatory sequences, for example, 3'UTR; and enhancers, etc. Mouse IL-15 is located on chromosome 8, NC_000074.7 (c83129883-83058253), and the mouse IL-15 coding sequence can be found in Genbank accession number NM_008357.3 (transcript variant 1); NM_001254747.2 (transcript variant 2). The regulatory sequences of mouse IL-15 are well defined in the art and can be readily identified using computer methods, for example, by reference to the above-mentioned Genbank accession numbers on the UCSC genome browser on the World Wide Web at genome.ucsc.edu, or by experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human IL-15 protein is located at the mouse IL-15 genomic locus, the regulatory sequences operably linked to the human IL-15 coding sequence are endogenous or native sequences of the mouse genome, that is, they are present in the mouse genome prior to integration of the human nucleic acid sequence.

[0225] In some examples, humanized IL-15 non-human animals (e.g., mice) are produced by randomly integrating or inserting a human nucleic acid sequence encoding a human IL-15 protein (including a fragment as described above), i.e., a "human IL-15 nucleic acid sequence" or a "human IL-15 sequence", into the genome of a non-human animal. Typically, in such embodiments, the position of the nucleic acid sequence encoding the human IL-15 protein in the genome is unknown. In other cases, humanized IL-15 non-human animals are produced by targeted integration or insertion of a human IL-15 nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while removing host genomic material, for example, removing 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material from the target locus. Therefore, for example, in the humanized IL-15 mouse comprising the nucleic acid sequence of encoding human IL-15 protein created by targeting human IL-15 nucleic acid sequence to mouse IL-15 locus, human IL-15 nucleic acid sequence can replace part or all of mouse sequence at IL-15 locus, for example, exon and / or intron. In some such examples, human IL-15 nucleic acid sequence is integrated into mouse IL-15 locus so that the expression of human IL-15 sequence is regulated by the natural or endogenous regulatory sequence at mouse IL-15 locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding human IL-15 protein is operably connected is the natural IL-15 regulatory sequence at mouse IL-15 locus.

[0226] In some cases, the integration of human IL-15 sequence will not affect the transcription of the gene in which the human IL-15 sequence has been integrated. For example, if the human IL-15 sequence is integrated into the coding sequence as an intein, or the human IL-15 sequence includes a 2A peptide, the human IL-15 sequence will be transcribed and translated simultaneously with the gene in which the human IL-15 sequence has been integrated. In other cases, the integration of human IL-15 sequence interrupts the transcription of the gene in which the human IL-15 sequence has been integrated. For example, when the human IL-15 sequence is integrated by homologous recombination, some or all of the coding sequence at the integration site can be removed, thereby transcribing the human IL-15 sequence. In some such examples, the integration of human IL-15 sequence will produce invalid mutations, so invalid alleles will be produced. Invalid alleles are mutant copies of genes that completely lack the normal function of the gene. This can be due to the complete absence of gene products (proteins, RNA) or the expression of non-functional gene products at the molecular level. At the phenotypic level, invalid alleles are indistinguishable from the absence of the entire locus.

[0227] In some instances, humanized IL-15 non-human animals (e.g., mice) include a copy of the nucleic acid sequence encoding human IL-15 protein. For example, non-human animals can be heterozygous for nucleic acid sequences. In other words, an allele on a locus will include the nucleic acid sequence, and another allele will be an endogenous allele. For example, as described above, in some cases, human IL-15 nucleic acid sequences are integrated into non-human animal (e.g., mice) IL-15 locus, thereby producing the invalid allele of non-human animal IL-15. In some such embodiments, humanized IL-15 non-human animals can be heterozygous for the nucleic acid coding sequence encoding human IL-15, that is, humanized IL-15 non-human animals include an invalid allele (including the allele of the nucleic acid sequence) and an endogenous IL-15 allele (wild type or other) of a non-human animal IL-15. In other cases, humanized IL-15 includes two copies of the nucleic acid sequence encoding human IL-15 protein. For example, a non-human animal (e.g., a mouse) can be homozygous for a nucleic acid sequence, i.e., both alleles of one locus in the diploid genome will comprise the nucleic acid sequence, i.e., a humanized IL-15 non-human animal comprises two null alleles of non-human animal IL-15 (alleles comprising the nucleic acid sequence).

[0228] Although embodiments using human IL-15 genes in mice are discussed extensively herein, other non-human animals (eg, rodents, eg, rats) comprising human IL-15 genes are also provided.

[0229] WO 2016 / 168212 describes human IL-15 polypeptides, loci encoding human IL-15 polypeptides, and non-human animals expressing human IL-15 polypeptides, and the application is incorporated herein by reference.

[0230] Humanized liver

[0231] In some aspects, the transgenic non-human animals provided herein also include modifications to the animal's immune system so that it cannot produce an immune response to xenotransplanted cells (e.g., human hepatocytes) or its ability to produce an immune response to xenotransplanted cells (e.g., human hepatocytes) is reduced (e.g., the non-human animal is immunodeficient). In some cases, the animal is suitable for xenotransplantation of hepatocytes. In some cases, the animal is a transgenic non-human animal, wherein (1) non-human (i.e., endogenous) hepatocytes in the liver can be selectively and conditionally ablated; and (2) the immune system of the non-human animal has been modified so that it cannot produce an immune response to xenotransplanted cells (e.g., human hepatocytes) (e.g., the non-human animal is immunodeficient). Cells and genomes containing genetic modifications disclosed herein are also provided. Transgenic non-human animals disclosed herein can be used for in vivo implantation and expansion of xenotransplanted hepatocytes (e.g., human hepatocytes). Xenotransplantation refers to the transplantation of living cells, tissues, or organs from one species to another species. Such cells, tissues, or organs are called xenografts or xenotransplants (e.g., xenotransplanted cells).

[0232] Any suitable immunodeficient non-human animal can be used. For example, see Weber et al., (2009) Liver Transplantation 15:7-14, the entire contents of which are incorporated herein by reference for all purposes. Such non-human animals can be immunocompromised so that natural non-human T cells and B cells do not develop. For example, such immunodeficient non-human animals can lack functional natural non-human T cells, B cells and / or natural killer (NK) cells. An immunodeficient non-human animal refers to a non-human animal that lacks at least one basic function of the immune system. For example, an immunodeficient non-human animal refers to an animal that lacks a specific component of the immune system or lacks the function of a specific component of the immune system (such as, for example, natural non-human B cells, T cells or NK cells). In some cases, the immunodeficient animal lacks natural non-human macrophages. In some cases, the immunodeficient animal contains one or more genetic changes that prevent or inhibit the development of functional immune cells (such as natural non-human B cells, T cells or NK cells). Various immunodeficient animal models are known in the art, such as animals with genetic changes in the Rag gene (for example, the immunodeficient non-human animal is Rag1 - / - and / or Rag2 - / - ) and Il2rg gene (Il2rg - / -) (see Traggiai et al., (2004) Science, 304: 104, which is incorporated herein by reference); animals with severe combined immunodeficiency (SCID) mutations in the Prkdc gene (see, e.g., Mercer et al., (2001) Nat. Med. 7(8): 927-933, the entire contents of which are incorporated herein by reference for all purposes); animals with SCID mutations and inactivated endogenous Il2rg genes, such as in a NOG background (see, e.g., Ito et al., (2002) Blood 100(9): 3175-3182, the entire contents of which are incorporated herein by reference for all purposes); or animals with other mutations, such as X-linked SCID characterized by autosomal recessive SCID characterized by JAK3 mutations, ADA mutations, IL7R mutations, CD3 mutations, ARTEMIS (DCLRE1C) mutations, and CD45 (PTPRC) mutations. Although certain non-limiting examples of genetic alterations that result in immunodeficiency are provided above, other known immunodeficient non-human animals may also be used.

[0233] Any suitable genetic modification can be used to allow selective and conditional ablation of non-human (ie, endogenous) hepatocytes in the liver. In some cases, non-human animals contain an inactivated endogenous Fah gene. This inactivation leads to toxic accumulation of tyrosine catabolites in hepatocytes. Compound 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) can be used to block hydroxyphenylpyruvate dioxygenase upstream of FAH, thereby preventing the accumulation of hepatotoxic metabolites. In some cases, non-human animals contain a urokinase-type plasminogen activator gene (Plau; NCBI GeneID 18792 in mice) operably linked to a liver-specific promoter (such as an albumin promoter). See, e.g., Mercer et al., (2001) Nat. Med. 7 (8): 927-933, the entire contents of which are incorporated herein by reference for all purposes. Such non-human animals are hepatotoxic, leading to liver failure. In some cases, non-human animals include a herpes simplex virus type 1 thymidine kinase (HSVtk) gene, which is operably connected to a liver-specific promoter (such as an albumin promoter). See, for example, Hasegawa et al., (2011) Biochem.Biophys.Res.Commun.405 (3): 405-410, the entire contents of which are incorporated herein by reference for all purposes. Ganciclovir (GCV) is a drug that is nontoxic to human or mouse tissues, and its administration induces tissue-specific ablation of transgenic hepatocytes. Since HSVtk catalyzes GCV phosphorylation (which is a rate-limiting step that cannot be performed in mammalian cells lacking the transgene), the hepatocytes expressing the transgene are selectively destroyed. Although some non-limiting examples of genetic modifications that allow selective and conditional ablation of non-human (i.e., endogenous) hepatocytes in the liver are provided above, any other suitable genetic modifications may also be used.

[0234] In one example, a transgenic non-human animal for hepatocyte xenotransplantation (e.g., human hepatocyte transplantation) has the following inactivated genes (i.e., knocked out): Fah (encoding fumarylacetoacetase); Rag1 (encoding V(D)J recombination activating protein 1) and / or Rag2 (encoding V(D)J recombination activating protein 2); and Il2rg (encoding interleukin 2 receptor subunit γ). Also provided are transgenic non-human animal cells or genomes having the following inactivated genes (i.e., knocked out): Fah; Rag1 and / or Rag2; and Il2rg. In one example, the transgenic non-human animal is a rat. See, e.g., Carbonaro et al., (2022) Sci.Rep.12(1):14079 and US2016 / 0249591, the entire contents of each of which are incorporated herein by reference for all purposes. In another example, the transgenic non-human animal is a mouse. See, e.g., Strom et al., (2010) Methods Mol. Biol. 640: 491-509, Azuma et al., (2007) Nat. Biotechnol. 25(8): 903-910, and US 8,569,573, each of which is incorporated herein by reference in its entirety for all purposes. In another example, the transgenic non-human animal is a pig. See, e.g., US 9,000,257, the entirety of which is incorporated herein by reference for all purposes.

[0235] Fah is an important gene in the tyrosine catabolism pathway. When Fah mutates in animals, toxic intermediate metabolites accumulate in the liver, leading to hepatocyte loss and ultimately liver failure and death. This toxicity can be improved by blocking the activity of another tyrosine catabolism enzyme, 4-hydroxyphenylpyruvate dioxygenase, which can be achieved by administering the small molecule nitisinone (NTBC). When NTBC is administered, Fah mutant mice are healthy and energetic. However, when NTBC is withdrawn, they quickly become moribund and die. Therefore, the administration and withdrawal of NTBC allows precise temporal control of hepatocellular toxicity in non-human animals with Fah mutations.

[0236] Fumarylacetoacetase (also known as FAH, FAA, β-diketase, or fumarylacetoacetate hydrolase) is encoded by the Fah gene (also known as fumarylacetoacetate hydrolase). FAH is an enzyme required for the final step of the tyrosine catabolic pathway, hydrolyzing fumarylacetoacetate into fumarate and acetoacetate. Fah deficiency leads to the accumulation of toxic metabolites, including fumarylacetoacetate and maleylacetoacetate. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (nitisinone or NTBC) acts as an inhibitor of 4-hydroxyphenylpyruvate dioxygenase by blocking the accumulation of toxic metabolites such as fumarylacetoacetate and maleylacetoacetate and is effective in ameliorating liver and kidney damage in human patients with Fah deficiency. Several Fah mutations have been found to cause human type I tyrosinemia (hereditary tyrosinemia type 1, or HT). This condition is characterized by severe liver and kidney disease, neurological problems, and other signs and symptoms that begin in infancy. The altered Fah gene that causes this condition produces an unstable or inactive enzyme, resulting in reduced or absent fumarylacetoacetate hydrolase activity. Without sufficient fumarylacetoacetate hydrolase activity, tyrosine and its byproducts cannot be broken down properly. As a result, fumarylacetoacetate accumulates in the liver and kidneys. Elevated levels of fumarylacetoacetate are considered toxic to cells, and accumulation of this substance may lead to liver and kidney problems and other features of tyrosinemia type I.

[0237] Mouse Fah is mapped to 7D3 on chromosome 7; 7 48.36 cM (NCBI RefSeq Gene ID 14085; assembly GRCm39 (GCF_000001635.27); position NC_000073.7 (84234367..84255150, complement). References to the mouse Fah gene include the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type mouse FAH protein has been assigned UniProt accession number P35505 and NCBI accession number NP_034306.2. References to mouse FAH proteins include the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform has been assigned NCBI accession number NM_010176.4. References to mouse Fah mRNA (cDNA) and coding sequences include the classical wild-type form as well as all allelic forms and isoforms.

[0238] Rat Fah is mapped to 1q31 on chromosome 1 (NCBI RefSeq Gene ID 29383; assembly mRatBN7.2 (GCF_015227675.2); position NC_051336.1 (138548830..138571599, complement). References to the rat Fah gene include the classical wild-type form as well as all allelic forms and isoforms. The classical wild-type rat FAH protein has been assigned UniProt accession number P25093 and NCBI accession number NP_058877.1. References to rat FAH proteins include the classical wild-type form as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the classical isoform has been assigned NCBI accession number NM_017181.2. References to rat Fah mRNA (cDNA) and coding sequences include the classical wild-type form as well as all allelic forms and isoforms.

[0239] An inactivated endogenous Fah gene is a Fah gene that does not produce FAH protein or does not produce functional FAH protein. A non-human animal (or cell or genome) may contain an inactivated Fah gene in its germline. A non-human animal (or cell or genome) may be homozygous for an inactivating mutation in a Fah gene. As an example, an inactivated endogenous Fah gene may include an insertion, deletion, or one or more point mutations in an endogenous Fah gene, resulting in a lack of functional FAH protein expression. Some inactivated endogenous Fah genes may include the deletion or destruction of all endogenous Fah genes, or may include the deletion or destruction of an endogenous Fah gene fragment (i.e., a portion of a gene or a portion of a gene). For example, some, most, or all of the coding sequences in an endogenous Fah gene may be deleted or destroyed. In one example, the 5' fragment of a Fah gene may be deleted or destroyed (e.g., including a start codon). As an example, the inactivated endogenous Fah gene can be a gene in which the start codon of the endogenous Fah gene has been deleted or destroyed or mutated so that the start codon no longer works. For example, the start codon can be destroyed by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, replacing one or more nucleotides. In another example, the 3' fragment of the Fah gene can be deleted or destroyed (for example, including a stop codon). In another example, the internal fragment of the Fah gene (that is, a fragment from the middle of the Fah gene) can be deleted or destroyed. In another example, all coding sequences in the endogenous Fah gene are deleted or destroyed.

[0240] US2016 / 024959, US Patent No. 8,569,573, Azuma et al., Nature Biotechnology 25(8):903-10 (2007), Carbonaro et al., Scientific Reports 12:14079-89 (2022), Carbonaro et al., Sci. Adv. 9, eadf4490 (2023), and Wilson et al., Stem Cell Research 13:404-412 (2014) describe non-human animals with inactivated endogenous Fah genes, and non-human animals expressing Fah defects, and these documents are incorporated herein by reference.

[0241] Transgenic non-human animals containing various modifications described herein, such as Rag 2 (and optionally Rag1) knockout, Il2rg KO, Hmox-1 KO, Fah KO, and genetic humanization including Sirpa and other genes (such as, for example, M-CSF, CD47, etc. described herein), can be bred together, transplanted with human HSCs and human hepatocytes, and used as models for studying human parasitic infections (e.g., malaria) with liver and blood stages.

[0242] Transgenic non-human animals and ES cells

[0243] In certain aspects, provided herein are transgenic non-human animals (e.g., rodents such as rats or mice) comprising: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally a Fah gene knockout and / or one or more humanized loci disclosed herein, and transgenic non-human animal ES cells that can be used to make such non-human animals.

[0244] In certain aspects, provided herein are transgenic non-human animals (e.g., rodents, such as rats or mice) and non-human animals (e.g., rodents, such as rats or mice) ES cells, comprising in their germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more engineered loci described herein. In some embodiments, provided herein are transgenic non-human animals (e.g., mice) and non-human animals (e.g., mice) ES cells, comprising in their germline and / or genome: (i) Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more engineered loci described herein. In some embodiments, transgenic non-human animals (e.g., rats) and non-human animal (e.g., rat) ES cells are provided herein, comprising in their germline and / or genome: (i) Rag1 gene knockout; (ii) Rag2 gene knockout; (iii) IL2rg gene knockout; and (iv) homozygous null mutations in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more engineered loci described herein. For example, in some embodiments, non-human animals or ES cells comprise a humanized Sirpa locus provided herein in their germline and / or genome. In some embodiments, non-human animals or ES cells comprise a CD47 locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise an M-CSF locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise a GM-CSF locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells comprise a TPO locus provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells include EPO loci provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells include IL-3 loci provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells include IL-15 loci provided herein in their germline and / or genome. In certain embodiments, non-human animals or ES cells include inactivation (e.g., deletion) of Fah genes provided herein in their germline and / or genome. In some embodiments, non-human animals or ES cells are heterozygous for one or more loci, e.g., transgenic loci provided herein. In some embodiments, non-human animals or ES cells are homozygous for one or more loci provided herein, e.g., transgenic loci.

[0245] In some embodiments, non-human animal can be any non-human animal. In some embodiments, non-human animal is a vertebrate. In some embodiments, non-human animal is a mammal. In some embodiments, transgenic non-human animal described herein can be selected from the group consisting of mouse, rat, rabbit, pig, cattle (e.g., cow, bull, buffalo), deer, sheep, goat, llama, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For non-human animals of suitable transgenic ES cells that are not easily available, other methods can be used to manufacture transgenic non-human animals described herein. These methods include, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and using nuclear transfer to transfer the modified genome to suitable cells, such as oocytes, and incubating modified cells (e.g., modified oocytes) in non-human animals under appropriate conditions to form embryos.

[0246] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal, for example, Dipodoidea superfamily or Muroidea superfamily. In some embodiments, the non-human animal is a rodent. In certain embodiments, the rodent is a mouse, a rat or a hamster. In some embodiments, the rodent is selected from Muroidea superfamily. In some embodiments, the non-human animal is from a family selected from the following: Cricetidae (e.g., hamsters similar to mice), Cricetidae (e.g., hamsters, New World mice, voles), Muridae (e.g., true mice and rats, gerbils, spiny mice, bristle mice), Isla Muscicapidae (e.g., climbing mice, rock climbing mice, white-tailed mice, Madagascar rats and mice), Spiny Mountain Myrmice (e.g., spiny dormice) and Zokoridae (e.g., mole mice, bamboo rats and zokor). In some embodiments, rodents are selected from true mice or rats (Muridae), gerbils, spiny mice and bristle mice. In some embodiments, the mouse is from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from a mouse and a rat. In some embodiments, the non-human animal is a mouse.

[0247] In some embodiments, the non-human animal is a C57BL strain mouse. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the non-human animal is a 129 strain mouse. In some embodiments, the 129 strain is selected from the group consisting of the following: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. In some embodiments, the transgenic mouse is a mixed strain of the 129 strain and the C57BL strain. In some embodiments, the mouse is a mixed strain of the 129 strain and / or a mixed strain of the C57BL / 6 strain. In some embodiments, the 129 strain of the mixed strain is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mouse is a mixed strain of the BALB strain and another strain (eg, C57BL strain and / or 129 strain). In some embodiments, the non-human animal provided herein can be a mouse derived from any combination of the aforementioned strains.

[0248] In some embodiments, the non-human animal provided herein is a rat. In some embodiments, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6 and Dark Agouti. In some embodiments, the rat strain is a mixed strain of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0249] In certain embodiments, the transgenic non-human animal or ES cell comprises a plurality of loci provided herein in its genome and / or germline, such as a plurality of genetically engineered loci provided herein. For example, in some embodiments, the non-human animal or ES cell comprises in its germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the non-human animal or ES cell comprises in its germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in a non-human animal heme oxygenase-1 (Hmox-1) gene, and a humanized Sirpa locus provided herein. In some embodiments, the non-human animal or ES cell comprises in its germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, a humanized Sirpa locus provided herein, and a humanized M-CSF locus provided herein. In some embodiments, the non-human animal or ES cell comprises in its germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, a humanized Sirpa locus provided herein, a humanized M-CSF locus provided herein, and a humanized CD47 locus provided herein. In some embodiments, the non-human animal or ES cell comprises in its germline and / or genome: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, a humanized Sirpa locus provided herein, and optionally selected from a humanized CD47 locus provided herein, a humanized M-CSF locus provided herein, a humanized GM-CSF locus provided herein, a humanized TPO locus provided herein, a humanized EPO locus provided herein, a humanized IL-3 locus provided herein, a humanized IL-15 locus provided herein, and one or more humanized loci of the group consisting of any combination thereof. In some embodiments, the non-human animal or ES cell described herein further comprises inactivation (e.g., deletion) of the Fah gene in its germline and / or genome.

[0250] In certain aspects, the transgenic non-human animal does not express the Hmox-1 polypeptide. In certain aspects, the transgenic non-human animal expresses one or more human or humanized polypeptides encoded by the humanized loci provided herein. For example, in some embodiments, the non-human animal expresses a human or humanized Sirpa polypeptide. In certain embodiments, the non-human animal expresses a human or humanized CD47 polypeptide. In certain embodiments, the non-human animal expresses a human or humanized M-CSF polypeptide. In certain embodiments, the non-human animal expresses a human or humanized GM-CSF polypeptide. In certain embodiments, the non-human animal expresses a human or humanized TPO polypeptide. In certain embodiments, the non-human animal expresses a human or humanized EPO polypeptide. In certain embodiments, the non-human animal or ES cells of the non-human animal express a human or humanized IL-3 polypeptide. In certain embodiments, the non-human animal expresses a human or humanized IL-15 polypeptide. In certain embodiments, the non-human animal or ES cell does not express FAH protein. In certain embodiments, the transgenic non-human animal does not express Hmox-1 protein and FAH protein.

[0251] Transgenic non-human animals and ES cells can be generated using any suitable method known in the art. For example, such transgenic non-human animal ES cells can be generated using The technology is generated, which is described in U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, and Valenzuela et al., (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis" Nat. Biotech. 21 (6): 652-659, each of which is incorporated herein by reference. Genome-targeted nuclease systems can also be used for modification, such as CRISPR / Cas systems, transcription activator-like effector nucleases (TALEN) systems, or zinc finger nucleases (ZFN) systems. In some embodiments, CRISPR / Cas systems are used for modification, for example, as described in U.S. Patent Application Nos. 14 / 314,866, 14 / 515,503, 14 / 747,461, and 14 / 731,914, each of which is incorporated by reference. Transgenic rat ES cells and rats can be prepared according to US2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al., (2010) Nature 467:211-215, and Tong et al., (2011) Nat Protoc. 6 (6): doi: 10.1038 / nprot.2011.338 (all of the entire contents of these documents are incorporated herein by reference). Example 1 also provides an exemplary method for preparing such transgenic non-human animals and ES cells.

[0252] The ES cells described herein can then be used to generate non-human animals using methods known in the art. For example, the mouse non-human animal ES cells described herein can be used to generate Methods Transgenic mice are generated as described in US Pat. No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is incorporated herein by reference. The resulting mice can be bred to homozygosity.

[0253] Methods for preparing transgenic non-human animals and ES cells

[0254] In certain aspects, provided herein are methods for preparing non-human animals (e.g., mice or rats) and ES cells, the non-human animals and ES cells comprising one or more transgenic loci provided herein. For example, in some embodiments, provided herein are methods for preparing non-human animals (e.g., mice or rats) and ES cells, the non-human animals and ES cells comprising: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) homozygous null mutations in non-human animal heme oxygenase-1 (Hmox-1) genes. In some embodiments, provided herein are methods for preparing non-human animals (e.g., mice or rats) and ES cells, the non-human animals and ES cells comprising: (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) homozygous null mutations in non-human animal heme oxygenase-1 (Hmox-1) genes, and further comprising a humanized Sirpa locus provided herein. In some embodiments, provided herein are methods for preparing non-human animals (e.g., mice or rats) and ES cells, the non-human animals and ES cells also comprising a humanized CD47 locus provided herein, a humanized M-CSF locus provided herein, a humanized GM-CSF locus provided herein, a humanized TPO locus provided herein, a humanized EPO locus provided herein, a humanized IL-3 locus provided herein, and / or a humanized IL-15 locus provided herein. In some embodiments, provided herein are methods for preparing non-human animals (e.g., mice or rats) and ES cells, the non-human animals and ES cells comprising homozygous null mutations in the Fah gene. Exemplary methods for preparing transgenic non-human animals and ES cells provided herein are described in the description, examples, and / or accompanying drawings herein. The generation of non-human animals comprising null mutations in the Hmox-1 gene of non-human animals can be accomplished using any convenient method for preparing transgenic animals, for example, as known in the art or as described in Example 1 herein.

[0255] Production of non-human animals comprising a nucleic acid sequence encoding a human or humanized protein (e.g., hSIRPA, hCD47, hM-CSF, hGM-CSF, hTPO, hEPO, hIL-3, or hIL-15) can be accomplished using any convenient method for making transgenic animals, e.g., as known in the art or as described herein.

[0256] For example, nucleic acids encoding human or humanized proteins (e.g., hSIRPA, hCD47, hM-CSF, hGM-CSF, hTPO, hEPO, hIL-3, and / or hIL-15) can be incorporated into a recombinant vector in a form suitable for insertion into the host cell genome and expression of the human protein in a non-human host cell. In various embodiments, the recombinant vector can include one or more regulatory sequences that are operably linked to the nucleic acid encoding the human protein in a manner that allows the nucleic acid to be transcribed into mRNA and the mRNA to be translated into the human protein, as described above. It will be appreciated that the design of the vector can depend on factors such as the selection of the host cell to be transfected and / or the amount of human protein to be expressed.

[0257] The human nucleic acid sequence can then be introduced into animal cells using any of a variety of methods to produce transgenic animals expressing human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, transformation of embryonic stem cells, homologous recombination, and knock-in techniques. Methods for generating transgenic animals that can be used include, but are not limited to, Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol, Humana Press), Meyer et al., (2010, Proc. Nat. Acad. Sci. USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2nd Edition). ed. Sunderland, Massachusetts: Sinauer), U.S. Pat. No. 6,586,251, Rathinam et al., (2011, Blood 118:3119-28), Willinger et al., (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al., (2011, Proc Natl Acad Sci USA, 108:2378-83) and Valenzuela et al., (2003, Nat Biot 21:652-659).

[0258] For example, the transgenic animals of the theme can be created by introducing nucleic acid encoding human protein into oocytes, for example, by microinjection, and allowing oocytes to grow in female foster animals. In a preferred embodiment, the expression construct is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulated females the second day after mating, and injected with expression constructs. Injected oocytes are either cultured overnight, or directly transferred to the oviduct of 0.5 day pc pseudopregnant females. Superovulation, harvesting oocytes, expression construct injections and embryo transfer methods are known in the art, and described in Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Can be assessed by DNA analysis (such as PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (such as, ELISA, Western blotting, etc.) to assess whether there is introduced nucleic acid in offspring.

[0259] As another example, the construct comprising the nucleic acid sequence encoding human protein can be transfected into stem cells (e.g., ES cells or iPS cells) using well-known methods, such as electroporation, calcium phosphate precipitation, lipofection, etc. It can be assessed by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.) whether the cell is introduced with the nucleic acid. The cell determined to have been incorporated with the expression construct can then be introduced into the preimplantation embryo. For a detailed description of the method for the composition and method of the present invention known in the art, see Nagy et al., (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al., (1990, Development 110: 815-821), U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and Kraus et al., (2010, Genesis 48: 394-399).

[0260] In addition, as described in some of the following examples, Genetic engineering techniques are used to construct nucleic acid constructs (see, e.g., Valenzuela et al., (2003) High throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-59 and U.S. Pat. No. 6,586,251), introduced into stem cells (e.g., ES cells), and correctly targeted clones determined using allele loss and allele gain assays (Valenzuela et al., supra); correctly targeted ES cells can be used as donor ES cells, using Methods are introduced into 8-cell stage mouse embryos (see, e.g., U.S. Patent No. 7,294,754 and Poueymirou et al., 2007, F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1): 91-99). In addition, transgenic rat ES cells and rats can be prepared according to US2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), US2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al., (2010) Nature 467: 211-215, and Tong et al., (2011) Nat Protoc. 6(6): doi: 10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety).

[0261] In some embodiments, the transgenic founder animal can be mated with other animals carrying one or more genetic modifications. For example, the HMOX-1-deficient non-human animals provided herein can be further mated with other transgenic non-human animals carrying other genetic modifications, including the introduction of fully human or humanized genes, for example, hSirpa knock-in mice, hM-CSF knock-in mice, hCD47 knock-in mice, hIL-3 knock-in mice, hGM-CSF knock-in mice, hTPO knock-in mice, hEPO knock-in mice, hIL-15 knock-in mice, etc., or mated with gene knockout animals, for example, non-human animals lacking one or more proteins, for example, not expressing one or more genes thereof, for example, Rag1-deficient animals, Rag2-deficient animals, I12rg-deficient animals or FAH-deficient animals.

[0262] In another embodiment, stem cells, such as ES cells, can be generated such that they contain multiple genetic modifications, such as humanization or gene deletions as described herein, and such stem cells can be introduced into embryos to produce transgenic animals with several genetic modifications.

[0263] As described above, in some embodiments, the transgenic non-human animal is an immunodeficient animal. Transgenic non-human animals that are immunodeficient and contain one or more human or humanized proteins, such as hSIRPA, hIL-3, hGM-CSF, hM-CSF, hEPO, hCD47, hIL-15 and / or hTPO, can be produced using any convenient method for producing transgenic animals, such as methods known in the art or described herein. For example, the production of transgenic immunodeficient animals can be achieved by introducing nucleic acids encoding human proteins into oocytes or stem cells containing mutated SCID gene alleles or Rag and / or Rag2 and Il2rg null alleles, which, when homozygous, will result in immunodeficiency, as described above and in more detail in the working examples herein. Then, mice are produced using modified oocytes or ES cells using methods such as described herein and known in the art, and mated to produce immunodeficient mice containing the desired genetic modifications. As another example, a transgenic non-human animal can be generated in an immunocompetent background and crossed with an animal containing a mutant gene allele that, when hemizygous or homozygous, results in immunodeficiency, and the offspring mated to produce an immunodeficient animal that expresses at least one human protein of interest.

[0264] In some embodiments, the transgenic mice are treated to eliminate endogenous hematopoietic cells that may be present in the mice. In one embodiment, the treatment comprises irradiating the transgenic mice. In a specific embodiment, the newborn transgenic mice are irradiated with sublethal doses. In a specific embodiment, the newborns are irradiated with 2 x 200 cGy at 4 hour intervals.

[0265] Various embodiments of the present invention provide transgenic animals that contain human nucleic acid in substantially all of their cells, and transgenic animals that contain human nucleic acid in some cells but not all of their cells. In some cases, such as targeted recombination, a copy of the human nucleic acid will be integrated into the genome of the transgenic animal. In other cases, such as random integration, multiple copies of the human nucleic acid that are adjacent to each other or far apart can be integrated into the genome of the transgenic animal.

[0266] Thus, in some embodiments, the transgenic non-human animal of the subject matter can be an immunodeficient animal comprising a genome comprising a nucleic acid encoding a human polypeptide, the nucleic acid being operably linked to a corresponding non-human animal promoter, wherein the animal expresses the encoded human polypeptide. In other words, the transgenic immunodeficient non-human animal of the subject matter comprises a genome comprising a nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operably linked to a corresponding non-human promoter and polyadenylation signal, and wherein the animal expresses the encoded human polypeptide.

[0267] Other methods for producing transgenic non-human animals are described in U.S. Pat. No. US11019810, U.S. Patent Publication No. US2021 / 0161112, WO 2011 / 044050, WO 2012 / 112544, WO 2014 / 039782, WO 2014 / 071397, WO2015 / 179317 and WO 2016 / 168212 (each of which is incorporated herein by reference), wherein the transgenic non-human animal comprises a genome comprising nucleic acids encoding one or more human proteins, such as hSIRPA, hIL-3, hGM-CSF, hM-CSF, hEPO, hCD47, hIL-15 and / or hTPO.

[0268] Implantation

[0269] In some embodiments, the transgenic non-human animals of the subject matter also have immunodeficiency. "Immunodeficiency" includes defects in one or more aspects of the natural or endogenous immune system of the animal, for example, the animal lacks one or more types of functional host immune cells, for example, defects in the number and / or function of non-human B cells, the number and / or function of non-human T cells, the number and / or function of non-human NK cells.

[0270] One method of achieving immunodeficiency in a subject animal is by sublethal irradiation. Alternatively or additionally, immunodeficiency can be achieved by any of a number of genetic mutations known in the art, any of which can be bred alone or in combination into the subject transgenic non-human animals of the present disclosure, or can be used as a source of stem cells into which the genetic modifications disclosed herein can be introduced. Non-limiting examples include X-linked SCID associated with IL2RG gene mutations, characterized by a lymphocytic phenotype of T(-)B(+)NK(-); autosomal recessive SCID associated with Jak3 gene mutations, characterized by a lymphocytic phenotype of T(-)B(+)NK(-); ADA gene mutations characterized by a lymphocytic phenotype of T(-)B(-)NK(-); IL-7Rα chain mutations characterized by a lymphocytic phenotype of T(-)B(+)NK(+); CD3δ or ε mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); RAG1 and RAG2 mutations characterized by lymphocyte phenotype T(-)B(-)NK(+); Artemis gene mutations characterized by lymphocyte phenotype T(-)B(-)NK(+), CD45 gene mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); and Prkdcscld mutations characterized by lymphocyte phenotype T(-), B(-). Therefore, in some embodiments, the transgenic immunodeficient non-human animal has one or more defects selected from the following: IL2 receptor γ chain defect, Jak3 defect, ADA defect, IL7R defect, CD3 defect, RAG1 and / or RAG2 defect, Artemis defect, CD45 defect and Prkdc defect. In one embodiment, the immune deficiency is achieved by gene mutation or deletion in Rag1 and / or Rag2 and Il2rg genes. These and other immunodeficient animal models will be known to those of ordinary skill in the art, any of which can be used to generate the immunodeficient animals of the present disclosure.

[0271] In some embodiments, transgenic non-human animals according to the present invention find use as recipients of human hematopoietic cells capable of developing human immune cells from implanted human hematopoietic cells. Thus, in certain aspects of the present invention, the subject transgenic animals are transgenic immunodeficient non-human animals transplanted with human hematopoietic cells.

[0272] Any source of human hematopoietic stem cells, human hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitor cells (HSPCs) known in the art or described herein can be transplanted into transgenic immunodeficient non-human animals of the present disclosure. A suitable source of human hematopoietic cells known in the art is human umbilical cord blood cells, particularly CD34 positive (CD34+) cells. Another source of human hematopoietic cells is human fetal liver. Another source is human bone marrow. Also included are induced pluripotent stem cells (iHSCs) and induced hematopoietic stem cells (iHSCs) produced by the dedifferentiation of somatic cells, for example, by methods known in the art. Methods for transplanting human cells into non-human animals are fully described in the art and elsewhere herein, any of which can be used by ordinary technicians to achieve non-human animals implanted with the subject transgene.

[0273] Cell populations of particular interest include those containing hematopoietic stem or progenitor cells that will contribute to or reconstitute the hematopoietic system of the transgenic non-human animal, for example, peripheral blood leukocytes, fetal liver cells, fetal bone, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and purified hematopoietic stem cells, such as mobilized HSCs or umbilical cord blood HSCs.

[0274] The cell can be from any mammalian species, such as mice, rodents, dogs, cats, horses, cattle, sheep, primates, people, etc. In one embodiment, the cell is a human cell. The cell can be from an established cell line, or can be a primary cell, wherein "primary cell", "primary cell line" and "primary culture" can be used interchangeably herein to refer to cells and cell cultures derived from a subject and allowing the culture to grow in vitro for a limited number of passages (i.e., divisions). For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10 or 15 times, but does not have enough time to pass through the transition period. Typically, the primary cell line of the present disclosure maintains less than 10 passages in vitro.

[0275] If the cells are primary cells, they can be harvested from an individual by any convenient method. For example, cells (e.g., blood cells, such as leukocytes) can be harvested by apheresis, leukocyte apheresis, density gradient separation, etc. As another example, cells can be harvested by biopsy, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, gastric tissue, etc. Suitable solutions can be used to disperse or suspend harvested cells. This solution is typically a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., which is conveniently supplemented with fetal bovine serum or other naturally occurring factors, and combined with an acceptable low concentration buffer, typically 5-25mM. Convenient buffer includes HEPES, phosphate buffer, lactate buffer, etc.

[0276] In some cases, a heterogeneous cell population will be transplanted into a transgenic non-human animal. In other cases, a cell population enriched for a specific type of cell (e.g., progenitor cells, such as hematopoietic progenitor cells) will be implanted into a transgenic non-human animal. The cell population of interest can be enriched by any convenient separation technique. For example, the cells of interest can be enriched by a culture method. In this culture method, specific growth factors and nutrients are usually added to the culture to promote the survival and / or proliferation of a cell population relative to other cell populations. Other culture conditions that affect survival and / or proliferation include growth on an adherent or non-adherent matrix, culture for a specific length of time, etc. Such culture conditions are well known in the art. As another example, the cells of interest can be enriched by separating the cells of interest from the initial population by affinity separation techniques. Affinity separation techniques can include magnetic separation using magnetic beads coated with affinity reagents, affinity chromatography, "panning" using affinity reagents attached to a solid matrix (e.g., a plate), cytotoxic reagents connected to affinity reagents or used in combination with affinity reagents (e.g., complement and cytotoxins), or other convenient techniques. Technologies that provide accurate separation include fluorescence activated cell sorters, which vary in sophistication, such as multiple color channels, low angle and dull light scatter detection channels, impedance channels, etc. Cells can be screened for dead cells by using dyes associated with dead cells (e.g., propidium iodide). Any technique that does not unduly compromise the viability of the cells of interest can be used.

[0277] For example, using affinity separation technology, by contacting a population with an affinity reagent that specifically recognizes and selectively binds to a marker that is not expressed on the cell of interest, cells that are not cells of interest for transplantation can be depleted from the population. For example, in order to enrich a population of hematopoietic progenitor cells, cells expressing mature hematopoietic cell markers can be depleted. Additionally or alternatively, positive selection and separation can be performed by contacting a population with an affinity reagent that specifically recognizes and selectively binds to markers associated with hematopoietic progenitor cells (e.g., CD34, CD133, etc.). "Selective binding" refers to a molecule that preferentially binds to a target of interest, or that has a greater affinity for binding to a target than to binding to other molecules. For example, an antibody will bind to a molecule that contains an epitope that is specific to it, and will not bind to an irrelevant epitope. In some embodiments, an affinity reagent can be an antibody, i.e., an antibody that is specific to CD34, CD133, etc. In some embodiments, an affinity reagent can be a specific receptor or ligand of CD34, CD133, etc., e.g., a peptide ligand and a receptor; effector molecules and receptor molecules, T cell receptors that are specific to CD34, CD133, etc. In some embodiments, multiple affinity reagents specific for a marker of interest may be used.

[0278] Antibodies and T cell receptors found to be used as affinity reagents may be monoclonal or polyclonal and may be produced by transgenic animals, immunized animals, immortalized human or animal B cells, cells transfected with DNA vectors encoding antibodies or T cell receptors, and the like. The details of antibody preparation and their suitability as specific binding members are well known to those skilled in the art. Of particular interest is the use of labeled antibodies as affinity reagents. Conveniently, these antibodies are conjugated to a marker for separation. Tags include magnetic beads, allowing direct separation; biotin, which can be removed with avidin or streptavidin bound to a carrier; fluorescent dyes, which can be used with a fluorescence-activated cell sorter; or similar methods to facilitate the separation of specific cell types. Available fluorescent dyes include phycobiliproteins, such as phycoerythrin and allophycocyanin, fluorescein, and Texas Red. Typically, each antibody is labeled with a different fluorescent dye so that each marker can be sorted independently.

[0279] The initial cell population is contacted with the affinity reagent and incubated for a time sufficient to bind available cell surface antigens. The incubation is generally at least about 5 minutes, and generally less than about 60 minutes. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that the efficiency of the separation is not limited by a lack of antibody. The appropriate concentration is determined by titration, but typically the antibody is diluted to the volume of the cell suspension at about 1:50 (i.e., 1 part antibody to 50 parts reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about 1:500, about 1:1000, about 1:2000, or about 1:5000. The medium in which the cells are suspended will be any medium that maintains cell viability. A preferred medium is phosphate buffered saline containing 0.1% to 0.5% BSA or 1-4% goat serum. A variety of culture media are commercially available and can be used depending on the properties of the cells, including Dulbecco's modified Eagle's medium (dMEM), Hank's basic salt solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Iscove's medium, PBS containing 5 mM EDTA, etc., usually supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

[0280] The cells in the contacted population labeled with the affinity reagent can be selected by any convenient affinity separation technique, for example, as described above or known in the art. After separation, the separated cells can be collected in any appropriate culture medium that maintains cell viability, usually with a serum pad at the bottom of the collection tube. Various culture media are commercially available and can be used according to the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., usually supplemented with fetal bovine serum.

[0281] In this way, a composition highly enriched for a cell type of interest (e.g., hematopoietic cells) is achieved. These cells will account for about 70%, about 75%, about 80%, about 85%, about 90% or more of the cell composition, about 95% or more of the enriched cell composition, preferably about 95% or more of the enriched cell composition. In other words, the composition will be a substantially pure composition of the cells of interest.

[0282] The cells to be transplanted into transgenic non-human animals, whether heterogeneous cell populations or enriched cell populations, can be transplanted immediately. Alternatively, the cells can be frozen at liquid nitrogen temperatures and stored for a long time, thawed and can be reused. In this case, the cells are usually frozen in 10% DMSO, 50% serum, 40% buffered medium or other such solutions commonly used in the art to preserve the cells at such freezing temperatures and thawed in a manner commonly known in the art for thawing frozen cultured cells. Additionally or alternatively, the cells can be cultured in vitro under various culture conditions. The culture medium can be liquid or semisolid, for example containing agar, methylcellulose, etc. The cell population can be conveniently suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI-1640, usually supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol and antibiotics, such as penicillin and streptomycin. The culture can contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules capable of promoting cell survival, growth and / or differentiation in culture or in intact tissues through specific actions on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0283] Prior to transplantation into transgenic non-human animals, cells may be genetically modified, for example, to provide a selectable or traceable marker, to induce a genetic defect in the cell (e.g., for disease modeling), to repair a genetic defect or to ectopically express a gene in the cell (e.g., to determine whether such modification affects the course of the disease), etc. Cells may be genetically modified by transfection or transduction using a suitable vector, homologous recombination, or other appropriate techniques so that they express a gene of interest, or by using antisense mRNA, siRNA, or ribozymes to block the expression of undesired genes. Various techniques for introducing nucleic acids into target cells are known in the art. Various techniques may be employed to demonstrate that cells have been genetically modified. The genome of the cell may be restricted, whether or not it is amplified. Polymerase chain reaction; gel electrophoresis; restriction analysis; Southern, Northern, and Western blotting; sequencing; or similar techniques may all be employed. General methods of molecular and cellular biochemistry used for these and other purposes disclosed in this application can be found in standard textbooks, such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al., ed., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., ed., Academic Press 1999); Viral Vectors (Kaplift & Loewy, ed., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference. The reagents, cloning vectors and kits used for gene manipulation mentioned in this disclosure can be obtained from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.

[0284] The cells can be transplanted into the transgenic non-human animal by any convenient method, including, for example, intrahepatic injection, tail vein injection, retro-orbital injection, etc. Typically, about 0.5 x 10 5 -2x 10 6 pluripotent cells or progenitor cells, for example, about 1 x 10 5 -1x 10 6 cells, or approximately 2 x 10 5 -5x 10 5 In some cases, the mice are irradiated sublethally prior to transplantation of the human cells. In other words, the mice are exposed to sublethal doses of radiation, for example, as described in the Examples section below and as known in the art. The transplanted transgenic non-human animals are then maintained under laboratory animal housing conditions for at least 1 week, for example, 1 week or longer, or 2 weeks or longer, sometimes 4 weeks or longer, and in some cases 6 weeks or longer, to allow for adequate reconstitution of the immune system with the implanted cells.

[0285] In some embodiments, the transplanted human hematopoietic cells produce one or more implanted human cells in the transgenic non-human animal, selected from human CD34 positive cells, human hematopoietic stem cells, human hematopoietic cells, myeloid progenitor cells, erythroid progenitor cells, myeloid cells, dendritic cells, monocytes, neutrophils, mast cells, erythrocytes and combinations thereof. In one embodiment, the human human cells are present at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after implantation. In a specific embodiment, the human cells comprise erythroid cells.

[0286] In some embodiments, the transplanted human hematopoietic cells produce an implanted human blood lymphatic system in a transgenic non-human animal, the system comprising human hematopoietic stem cells and progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human erythrocytes, human thymocytes, human T cells, human B cells, and human platelets. In one embodiment, the human blood lymphatic system exists at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after implantation. In a specific embodiment, the human blood lymphatic system comprises erythroid cells.

[0287] Erythroid cells include red blood cells and cells that produce red blood cells. "Red blood cells" include mature red blood cells, also known as erythrocytes or erythrocytes. Cells that produce red blood cells include erythrocyte progenitor cells, i.e., multipotent cells that proliferate, and erythrocyte precursor cells, i.e., proliferating or non-proliferating cells that are committed to becoming red blood cells.

[0288] Red blood cells are the main cellular element in the circulating blood and have the transport of oxygen as their primary function. The number of red blood cells per cubic millimeter of blood is usually maintained between 4.5 and 5.5 million in men and between 4.2 and 4.8 million in women. It varies with age, activity, and environmental conditions. For example, at altitudes above 10,000 feet, it usually increases to a level of 8 million / mm. When it is removed from the bloodstream and broken down by the reticuloendothelial system, the red blood cell usually survives 110 to 120 days. New red blood cells are produced at a rate of slightly more than 1% per day; therefore, a constant level is usually maintained. Acute blood loss, hemolytic anemia, or chronic hypoxia may cause a large increase in erythropoiesis.

[0289] Erythrocytes originate from hematopoietic stem cells in the bone marrow of long bones and develop into erythrocytes through a series of cell stages, which include: common myeloid progenitor cells (CD123+, CD34+, c-kit+, Flt3+); megakaryocyte-erythrocyte progenitor cells (CD34+, CD38+, CD45RA-); proerythroblasts (if normal, also called proerythroblasts, if abnormal, called promegatrophytic erythroblasts; large CD71+, EpoR+, c-kit+, Terl 19+ progenitor cells); basophilic erythroblasts (cytoplasmic basophilia, in which the nucleus is large, chromatin is aggregated, and the nucleolus is effaced); polychromatic erythroblasts (also called mesoerythroblasts, in which the nuclear chromatin shows increased aggregation, and the cytoplasm begins to acquire hemoglobin and becomes eosinophilic); orthochromatic erythroblasts (the final stage before nuclear loss, in which the nucleus is small and eventually becomes a blue-black, uniform, structureless mass); and reticulocytes (circulating CD235+, CD71+ cells; cells are characterized by a reticular pattern of threads and granules in the more anterior part of the nucleus).

[0290] Mature erythrocytes appear as biconcave, round, or oval discs approximately 6–8 μm in diameter on peripheral blood smears. They contain hemoglobin and have a central pale area due to the double chamber of the cell and can be easily identified by flow cytometry or immunohistochemistry-based methods by elevated expression of the cell surface markers CD235 and CD59 relative to non-erythrocytes.

[0291] In certain aspects, the transgenic non-human animals provided herein are transplanted with human hematopoietic cells and include infections caused by human pathogens. Of particular interest in these embodiments are human pathogens that target human erythroid cells. Non-limiting examples of such pathogens include protozoa of the genus Plasmodium, Babesia, and Theileria, etc. As described in more detail below, the subject transgenic non-human animals transplanted with human hematopoietic cells can be infected with human pathogens using any suitable method of infecting animals with interested pathogens known in the art or described herein. Animals infected in this way will typically show signs of parasitemia, including morphological changes shown by blood smears stained with Giemsa, and severe decreases in total red blood cell concentration (e.g., 50%) and anemia.

[0292] In certain aspects, the transgenic non-human animals provided herein are transplanted with human hematopoietic cells comprising a disease-specific mutation, for example, a mutation in the β-globin gene that causes sickle cell disease.

[0293] Non-restrictive applications of transgenic transplant mice

[0294] Transgenic non-human animals disclosed herein have many uses in the art. For example, the transplanted transgenic animals disclosed herein can be used to study human erythropoiesis and the function of human erythrocytes. As another example, the transplanted transgenic mice disclosed herein provide a useful system for screening the desired activity in vivo of candidate agents, for example, identifying agents that can regulate (i.e., promote or inhibit) human erythropoiesis and / or human erythrocyte function, for example, in a healthy or diseased state (e.g., as cancer cells, during pathogen infection, etc.). For example, the transplanted transgenic mice disclosed herein can be used to identify new treatment methods; or as another example, identify agents that are toxic to human erythrocytes, and identify agents that prevent, reduce or reverse the toxic effects of toxic agents on human erythrocytes; etc. As another example, the transplanted transgenic animals disclosed herein provide a useful system for predicting the responsiveness of individuals to disease therapies, for example, by providing an in vivo platform to screen the responsiveness of an individual's immune system to an agent (e.g., a therapeutic agent) to predict the responsiveness of an individual to the agent. As yet another example, hematopoietic stem and progenitor cells (HSPCs) from patients with genetic abnormalities of red blood cells (e.g., sickle cell anemia, β-thalassemia, etc.) or iPS-derived HSPCs with such genetic modifications can be transplanted into HMOX-1-deficient HIS mice to model certain red blood cell diseases and test potential therapeutics.

[0295] As a non-limiting example, the transplanted transgenic mice of the present disclosure find use in generating mouse models of pathogen infection caused by parasites that target human erythrocytes, such as Plasmodium, Babesia, and Theileria, among others. Such mouse infection models can be used for research, e.g., to better understand the progression of human infection, and for drug discovery, e.g., to identify candidate agents for preventing or treating infections caused by such parasites.

[0296] Protozoa of the genus Plasmodium are the cause of malaria in humans. Malaria begins with the bite of an infected Anopheles mosquito, where the protozoa enter the circulatory system through their saliva and eventually travel to the liver, where they mature and multiply. The protozoa then enter the bloodstream and infect erythroid cells at various stages of maturation.

[0297] Five species of Plasmodium can infect and be transmitted by humans. The vast majority of deaths are caused by Plasmodium falciparum, while malaria caused by Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae is generally milder and rarely fatal. This is thought to be due, at least in part, to the cell types targeted by each species: Plasmodium falciparum grows in all mature red blood cells (RBCs), while Plasmodium vivax, for example, is restricted to growth in reticulocytes, which make up only about 1%-2% of the total red blood cells in peripheral blood. In addition, Plasmodium falciparum causes severe malaria through a unique property not shared by any other human malaria, namely, retention. During the 48-hour asexual blood stage cycle, the mature form changes the surface properties of infected red blood cells, causing them to adhere to blood vessels (a process called cell adhesion). This can cause microcirculatory obstruction and lead to multiple organ dysfunction.

[0298] Symptoms of malaria include fever, chills, headache, sweating, fatigue, anemia, nausea, dry cough (non-productive), muscle and / or back pain, and an enlarged spleen. Other symptoms and complications associated with malaria include brain infection (encephalitis), hemolytic anemia, kidney failure, liver failure, meningitis, pulmonary edema, and splenic bleeding. Generally, individuals at risk for malaria begin to develop symptoms 7 days or more after infection, for example, 9 to 14 days after a primary infection with Plasmodium falciparum, 12 to 18 days after a primary infection with Plasmodium vivax or Plasmodium ovale, 18 to 40 days after a primary infection with Plasmodium malariae, or 11 to 12 days after a primary infection with Plasmodium knowlesi. Antimalarial drugs used in the art to treat or prevent malaria include chloroquine, quinidine, doxycycline, tetracycline, clindamycin, atovaquone plus proguanil (Malarone), mefloquine, artesunate, and pyrimethamine plus sulfadoxine (Fansidar).

[0299] Methods for determining whether a subject has been infected with Plasmodium are recognized in the art and include, for example, microscopic examination of blood using blood films, use of antigen-based rapid diagnostic tests (RDTs), e.g., immunochromatographic-based RDTs, detection of parasite DNA by polymerase chain reaction (PCR), etc. Any convenient method may be used to determine whether a subject's human red blood cells are infected with a pathogen.

[0300] HMOX-1 in HSC transplantation - / - The presence of human erythrocytes in the peripheral blood of the human immune system (HIS) mouse model provides a unique opportunity to model malaria with an in vivo mouse model. Malaria begins a life cycle stage after infection, with sporozoites injected from the mosquito bite initially infecting the liver, and then the merozoites that emerge infect human erythrocytes (Vaughan et al. (2012) J Clin Invest 122(10):3618-28; Minkah et al. (2018) Front Immunol. 9:807; Kaushansky et al. (2014) Cell Microbiol. 16(5):602-11; Good et al. (2015) Trends Parasitol. 31(11):583-94; Foquet et al. (2017) Methods Mol Biol. 1506:117-30). Although human liver infection has been modeled in vivo using immunodeficient mice that can be transplanted with human hepatocytes, for example, in mice deficient in fumarylacetoacetate hydrolase (FAH), allowing ablation of mouse liver and replacement with human liver, in vivo studies in malaria mice have been hampered by the lack of survival of human erythrocytes in mice (Vaughan et al., (2012) J Clin Invest 122(10):3618-28). By combining the HMOX-1-deficient HIS model with modifications that allow engraftment of human livers, an excellent model has been generated for studying the entire malaria life cycle and testing potential therapeutics at all stages of malaria.

[0301] Thus, in one embodiment, the HMOX-1-deficient MSRG47 mice or rats described herein are bred to the FAH- / -SRG model (FSRG) (see, e.g., Carbonaro et al. (2023) Sci. Adv. 9, eadf4490; Carbonaro et al. (2022) Sci. Rep. 12: 14079, both incorporated herein by reference) to generate a human immune system model that allows human erythrocytes, human leukocytes, and human hepatocytes to develop (e.g., after human HSC and human hepatocyte engraftment). Such animals can be used to test potential malaria therapies, particularly immune-based therapies, and are contemplated for use in the methods described herein for screening potential therapies.

[0302] Another example of a pathogen of interest is a protozoan of the genus Babesia. Infection with Babesia causes a malaria-like disease called babesiosis. Babesiosis is a vector-borne disease, usually transmitted by the Ixodes scapularis tick. The disease is usually caused by Babesia muris in humans, Babesia rosenbergii subsp. and Babesia canis subsp. in dogs, Babesia bovis in dairy cows, and Babesia bovis in cattle. Babesia muris infects humans using the same tick vectors as Lyme disease and ehrlichiosis, and can occur in conjunction with these other diseases. The protozoan can also be transmitted through blood transfusions.

[0303] In humans, babesiosis can be asymptomatic or characterized by symptoms ranging from mild fever and diarrhea to high fever, chills, and severe anemia. In severe cases, organ failure may occur, including respiratory distress syndrome. Severe cases occur mostly in people who have undergone a splenectomy or who have immune deficiencies, such as those with HIV / AIDS. Treatment usually consists of a two-drug regimen of quinine and clindamycin or atovaquone and azithromycin. In life-threatening cases of babesiosis, blood exchange transfusions are performed in which infected red blood cells are removed and replaced with uninfected red blood cells.

[0304] Definitive diagnosis of Babesia infection is by identification of the parasite on a Giemsa-stained thin blood smear. The parasite appears as paired merozoites in erythrocytes, forming a "Maltese cross formation" in humans, or "two pears hanging together" in animals. Other diagnostic methods include peripheral blood PCR and serological testing for anti-Babesia antibodies (IgG, IgM).

[0305] Another malaria-like disease, Theileriosis, is caused by protozoa of the genus Theileria. In humans, Theileriosis is caused by T. microti; in horses, by T. equi ("equine piroplasmosis"); in sheep and goats, by T. lestoquardi; and in cattle, Cape buffalo, water buffalo, and eland, by T. annularis ("annular Theileriosis," also known as "Mediterranean Theileriosis") or T. parva ("East Coast fever," also known as "corridor disease"). Theileriosis is transmitted to hosts by various tick species, including Ixodes scapularis, Rhipicephalus, Cynocephalus, Haemaphysalis, and Hyalomma. The organism reproduces in the tick as it progresses through its life stages and matures and enters the saliva after the tick attaches to the host. Typically, a tick must be attached for several days to become infectious. However, if ambient temperatures are high, infective sporozoites can develop in ticks on the ground and may enter the host within hours of attachment.

[0306] In humans, theileriasis usually presents with fever and hemolysis. The definitive diagnosis of Theileria infection is by identification of the parasite on a Giemsa-stained thin blood smear.

[0307] Transgenic animals transplanted with the present disclosure are found to be useful for screening candidate agents to identify those that will prevent (e.g., vaccines) or treat infections caused by Plasmodium, Babesia, Theileria, and other parasites that target human erythrocytes. The term "treatment" and the like as used herein generally include obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, by preventing the disease or its symptoms in whole or in part, and / or may be therapeutic, by partially or completely curing the disease and / or adverse reactions caused by the disease. "Treatment" as used herein includes any treatment for a mammalian disease, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing the disease to regress. Candidate agents of interest as antiparasitic therapeutics include those that can be administered before, during, or after infection with a parasite and, when administered in an effective amount, inhibit the effects of the parasite on the individual (i.e., host), for example, by killing the parasite or cells infected by the parasite, by preventing the reproduction of the parasite, by preventing the production or effect of agents (i.e., toxins) produced by the parasite that are toxic to the individual, etc. The terms "individual," "subject," "host," and "patient" are used interchangeably herein and include any mammalian subject, particularly humans, in need of diagnosis, treatment, or therapy.

[0308] In the screening assay of bioactive agents, the transgenic non-human animal of the human hematopoietic cell transplantation disclosed herein is contacted with a candidate agent of interest, and the effect of the candidate agent is evaluated by monitoring one or more output parameters. These output parameters can reflect the vitality of human erythrocytes, for example, the total number of human erythrocytes; or the apoptotic state of human erythrocytes, for example, the amount of cell blistering, the amount of phosphatidylserine on the surface of human erythrocytes, etc., by methods well known in the art. Alternatively or additionally, the output parameters can reflect the effect of the agent on infection in the human erythrocytes of the transgenic non-human animal of the human hematopoietic cell transplantation disclosed herein.

[0309] The candidate agents or methods for screening disclosed herein may include, for example, organic molecules (e.g., small molecule inhibitors), nucleic acids (e.g., RNA interfering agents, oligonucleotides or nucleic acids encoding polypeptides), peptides, peptidomimetic inhibitors, aptamers, antibodies, intracellular antibodies, etc. As used herein, "RNA interfering agents" may be small interfering RNA (siRNA), CRISPR RNA (crRNA), CRISPR guide RNA (gRNA), small hairpin RNA (shRNA), micro RNA (miRNA) or piwi protein interacting RNA (piRNA).

[0310] By applying candidate agent to at least one sample, and usually multiple samples will be applied candidate agent, sometimes in conjunction with the sample lacking this candidate agent, to screen the biological activity of the candidate agent.Measurement is in response to the parameter change of the agent, and by comparing with reference sample to evaluate the result, for example, in the presence and absence of the agent, the result obtained using other agents, etc.Screen therein to identify the candidate agent that will prevent, alleviate or reverse the effect of pathogen, usually screen in the presence of pathogen, wherein pathogen is added at the time that is most suitable for the result to be determined.For example, in the case of the protection / prevention ability of testing candidate agent, candidate agent can be added before pathogen, add simultaneously with pathogen, or add after pathogen infection.As another example, in the case of the ability of testing candidate agent to reverse the effect of pathogen, candidate agent can be added after infecting pathogen.As mentioned above, in some cases, " sample " is the transgenic non-human animal that has implanted cell, for example, candidate agent is provided to the transgenic non-human animal that has implanted artificial hematopoietic cell. In some cases, the "sample" is a human hematopoietic cell to be transplanted, ie, the candidate agent is provided to the cells, such as reticulocytes, erythrocytes, etc., prior to implantation into an immunodeficient transgenic animal.

[0311] If the candidate agent is directly administered to the transplanted transgenic animal, the agent can be administered by any of the many well-known methods in the art for administering peptides, small molecules and nucleic acids to mice. For example, the agent can be administered orally, mucosally, topically, intradermally or by injection, such as intraperitoneally, subcutaneously, intramuscularly or intravenously. The agent can be administered in a buffer, or can be incorporated into any of the various preparations, for example, by using in combination with a suitable pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" can be a carrier approved by a federal or state government regulatory agency, or a carrier for mammals (such as humans) listed in the U.S. Pharmacopoeia or other recognized pharmacopoeias. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle, with which the compound of the present invention is formulated for administration to mammals. This pharmaceutical carrier can be a lipid, such as a liposome, such as a liposome dendrimer; a liquid, such as water and oil, including liquids of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can also be used. The pharmaceutical composition can be formulated into a preparation in the form of a solid, semisolid, liquid or gas, such as a tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere and aerosol. The agent can be systemic after administration, or can be focalized by using regional administration, intramural administration or using an implant that acts at the implantation site to maintain an active dose. The active agent can be formulated as an immediate activity, or can be formulated as a sustained release. If the agent is provided to the cell before implantation, the agent can be conveniently added to the culture medium of the cultured cell in a solution or soluble form. The agent can be added to the circulation system as a stream, intermittently or continuously, or alternatively, the compound mass is added individually or incrementally to an otherwise static solution. In the circulation system, two fluids are used, one of which is a physiologically neutral solution and the other is the same solution to which the test compound has been added. The first liquid flows through the cells, and then the second liquid flows through the cells. In the single solution method, the test compound mass is added to the culture medium volume surrounding the cells. The total concentration of the culture medium components should not change significantly with the addition of the compound mass, or change significantly between the two solutions in the circulation method.

[0312] Multiple determinations can be performed in parallel with different agent concentrations to obtain differential responses to different concentrations. As known in the art, the effective concentration of the agent is usually determined using a concentration range derived from a dilution of 1:10, or other logarithmic scale. If necessary, the concentration can be further optimized by a second series of dilutions. Typically, one of these concentrations is used as a negative control, that is, at zero concentration or below the detection level of the agent, or at or below the agent concentration that does not produce a detectable phenotypic change.

[0313] The response analysis of cells to candidate agents in the transgenic animals that can be transplanted at any time after the agent is treated. For example, cells can be analyzed 1, 2 or 3 days after contacting the candidate agent, sometimes 4, 5 or 6 days, sometimes 8, 9 or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or longer, for example, 2 months, 4 months, 6 months or longer. In some embodiments, the analysis includes the analysis of multiple time points. The selection of the analysis time point will be based on the analysis type to be performed, which is easily understood by those of ordinary skill.

[0314] The analysis may include measuring any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, particularly because they may be associated with cells of immune cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type. Histochemistry or immunohistochemistry can be performed to determine the apoptotic state of the cells, for example, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to measure DNA fragmentation, or immunohistochemistry to detect the binding of annexin V to phosphatidylserine on the cell surface. Flow cytometry can also be used to assess the ratio of differentiated cells and differentiated cell types, for example, to determine the ability of hematopoietic cells to survive and / or differentiate in the presence of an agent. ELISA, Western and Northern blotting can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in transplanted transgenic mice, for example, to assess the function of transplanted cells, assess the survival of erythrocytes, etc. In vivo assays can also be performed to test the function of immune cells, as well as assays associated with specific diseases or conditions of interest (such as anemia, such as sickle cell anemia, etc.). See, e.g., Current Protocols in Immunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), the disclosures of which are incorporated herein by reference.

[0315] Thus, for example, a method for determining the effect of an agent on red blood cells that can be infected or infected by a pathogen is provided, comprising administering the agent to a transgenic non-human animal of the present disclosure, the animal having been implanted with human reticulocytes and / or red blood cells; measuring the viability parameters of the transplanted cells over time in the presence of the agent; and comparing the measured values ​​with the measured values ​​of the transplanted transgenic non-human animal that has not been exposed to the agent. After one or more administrations of the agent within a selected time period, if the agent reduces the infection and / or death of human red blood cells in the peripheral blood of the mouse by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (i.e., an undetectable amount), the agent is determined to be anti-pathogen. In a specific embodiment, the administration of the drug or drug combination is at least 3 days, at least 1 week, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks after implantation of human hematopoietic cells.

[0316] Other examples for the subject mice are provided elsewhere herein.Other applications of the transgenic and transplanted mice described in this disclosure will be apparent to those skilled in the art reading this disclosure.

[0317] Additional exemplary embodiments

[0318] In exemplary embodiment 1, provided herein is a transgenic non-human animal comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0319] In exemplary embodiment 2, provided herein is the transgenic non-human animal of embodiment 1, wherein the transgenic non-human animal comprises a homozygous null mutation in the Rag1 gene.

[0320] In exemplary embodiment 3, provided herein is the transgenic non-human animal of embodiment 1 or 2, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

[0321] In exemplary embodiment 4, provided herein is the transgenic non-human animal of any one of embodiments 1-3, wherein the null mutation is a deletion of the entire endogenous coding sequence of Hmox-1.

[0322] In exemplary embodiment 5, provided herein is the transgenic non-human animal of any one of embodiments 1-4, comprising a homozygous null mutation in the Fah gene.

[0323] In exemplary embodiment 6, provided herein is the transgenic non-human animal of embodiment 5, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0324] In exemplary embodiment 7, provided herein is the transgenic non-human animal of any one of embodiments 1-6, wherein the transgenic non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0325] In exemplary embodiment 8, provided herein is the transgenic non-human animal of embodiment 7, wherein the transgenic non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0326] In exemplary embodiment 9, provided herein is the transgenic non-human animal of embodiment 8, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0327] In exemplary embodiment 10, provided herein is the transgenic non-human animal of embodiment 8 or 9, wherein the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

[0328] In exemplary embodiment 11, provided herein is the transgenic non-human animal of any one of embodiments 8-10, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0329] In exemplary embodiment 12, provided herein is the transgenic non-human animal of embodiment 7, wherein the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0330] In exemplary embodiment 13, provided herein is a transgenic non-human animal according to any one of embodiments 7-12, wherein the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0331] In exemplary embodiment 14, provided herein is a transgenic non-human animal of any one of embodiments 7-13, wherein the at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

[0332] In exemplary embodiment 15, provided herein is the transgenic non-human animal of embodiment 14, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0333] In exemplary embodiment 16, provided herein is the transgenic non-human animal of embodiment 14 or 15, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0334] In exemplary embodiment 17, provided herein is the transgenic non-human animal of any one of embodiments 7-16, comprising a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

[0335] In exemplary embodiment 18, provided herein is a transgenic non-human animal of any one of embodiments 7-17, wherein the transgenic non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0336] In exemplary embodiment 19, provided herein is a transgenic non-human animal of any one of embodiments 7-17, wherein the transgenic non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0337] In exemplary embodiment 20, provided herein is the transgenic non-human animal of any one of embodiments 7-19, wherein the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0338] In exemplary embodiment 21, provided herein is the transgenic non-human animal of any one of embodiments 7-20, wherein the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0339] In exemplary embodiment 22, provided herein is the transgenic non-human animal of embodiment 21, wherein the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0340] In exemplary embodiment 23, provided herein is a transgenic non-human animal according to any one of embodiments 7-22, wherein the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0341] In exemplary embodiment 24, provided herein is a transgenic non-human animal of any one of embodiments 7-23, wherein the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0342] In exemplary embodiment 25, provided herein is the transgenic non-human animal of any one of embodiments 1-24, further comprising an implant of human hematopoietic cells.

[0343] In exemplary embodiment 26, provided herein is the transgenic non-human animal of embodiment 25, wherein the human hematopoietic cells include one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human erythroid precursor cells, and human red blood cells.

[0344] In exemplary embodiment 27, provided herein is the transgenic non-human animal of embodiment 36, wherein the animal comprises human erythroid cells.

[0345] In exemplary embodiment 28, provided herein is the transgenic non-human animal of embodiment 27, wherein the non-human animal further comprises an infection with a pathogen that targets human erythroid cells.

[0346] In exemplary embodiment 29, provided herein is the transgenic non-human animal of any one of embodiments 25-28, wherein the animal comprises an inactivated endogenous FAH gene, and wherein the animal further comprises transplanted human hepatocytes.

[0347] In exemplary embodiment 30, provided herein is the transgenic non-human animal of embodiment 28 or embodiment 29, wherein the pathogen can cause human malaria.

[0348] In exemplary embodiment 31, provided herein is the transgenic non-human animal of embodiment 28, wherein the pathogen is selected from Plasmodium species, Babesia species, and Theileria species.

[0349] In exemplary embodiment 32, provided herein is the transgenic non-human animal of any one of embodiments 25-27, wherein the implanted human hematopoietic cells produce abnormal human erythroid cells.

[0350] In exemplary embodiment 33, provided herein is the transgenic non-human animal of embodiment 32, wherein the implanted human hematopoietic cells comprise a mutation in the β-globin gene that causes sickle cell disease.

[0351] In exemplary embodiment 34, provided herein is the transgenic non-human animal of any one of embodiments 1-33, wherein the transgenic non-human animal is a mammal.

[0352] In exemplary embodiment 35, provided herein is the transgenic non-human animal of embodiment 34, wherein the mammal is a rodent, such as a rat or a mouse.

[0353] In exemplary embodiment 36, provided herein is the transgenic non-human animal of embodiment 35, wherein the rodent is a mouse.

[0354] In exemplary embodiment 37, the present invention provides a method for identifying an agent that inhibits infection caused by a pathogen targeting human erythroid cells, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; iii. implantation of human hematopoietic cells; and iv. infection with a pathogen targeting human erythroid cells, and b. determining whether the agent reduces the amount of the pathogen and / or inhibits the activity of the pathogen in the non-human animal infected with the pathogen.

[0355] In exemplary embodiment 38, the present invention provides a method for identifying an agent that prevents infection by a pathogen that targets human erythrocytes, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic cells, b. injecting reticulocytes or red blood cells infected with parasites into the transgenic non-human animal, and c. determining whether the agent prevents infection of the non-human animal by the human reticulocytes and / or the red blood cells.

[0356] In exemplary embodiment 39, provided herein is the method of embodiment 37 or 38, wherein the pathogen can cause human malaria.

[0357] In exemplary embodiment 40, provided herein is the method of embodiment 37 or 38, wherein the pathogen is selected from Plasmodium species, Babesia species, and Theileria species.

[0358] In exemplary embodiment 41, a method for identifying an agent for treating sickle cell disease comprises: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic cells comprising a β-globin gene mutation that causes sickle cell disease, and b. determining whether the agent prevents or reduces sickling of red blood cells in the non-human animal.

[0359] In exemplary embodiment 42, the present invention provides a method for evaluating the therapeutic effect of a candidate drug targeting human red blood cells, the method comprising: a. administering the candidate drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous invalid mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous invalid mutation in the Rag2 gene and a homozygous invalid mutation in the IL2rg gene; and iii. implantation of human hematopoietic progenitor cells, and b. monitoring the human red blood cells in the non-human animal to evaluate the therapeutic effect of the candidate drug.

[0360] In exemplary embodiment 43, provided herein is a method of embodiment 42, wherein the human red blood cells are monitored to determine whether the candidate drug increases the production and / or survival of human red blood cells in the non-human animal.

[0361] In exemplary embodiment 44, the present invention provides a method for evaluating the toxicity of a candidate drug to human red blood cells, comprising: a. administering the candidate drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous invalid mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous invalid mutation in the Rag2 gene and a homozygous invalid mutation in the IL2rg gene; and iii. implantation of human hematopoietic progenitor cells, and b. monitoring the human red blood cells in the non-human animal to evaluate the toxicity of the candidate drug.

[0362] In exemplary embodiment 45, provided herein is the method of embodiment 44, wherein the toxicity is on-target toxicity or off-target toxicity.

[0363] In exemplary embodiment 46, provided herein is the method of embodiment 44 or 45, wherein the human red blood cells are monitored to determine whether the candidate drug reduces the number of human red blood cells in the non-human animal.

[0364] In exemplary embodiment 47, provided herein is the method of embodiment 46, wherein the candidate drug is a chemotherapeutic agent or an anti-malarial agent.

[0365] In exemplary embodiment 48, provided herein is the method of embodiment 44 or 45, wherein the human red blood cells are monitored to assess whether the candidate drug induces agglutination of the red blood cells.

[0366] In exemplary embodiment 49, provided herein is the method of embodiment 48, wherein the candidate drug is a modulator of human CD47 protein.

[0367] In exemplary embodiment 50, provided herein is the method of embodiment 49, wherein the modulator is an antibody.

[0368] In exemplary embodiment 51, the present invention provides a method for identifying an agent that reduces the toxicity of a toxic drug to human red blood cells, comprising: a. administering the agent and the toxic drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous invalid mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous invalid mutation in the Rag2 gene and a homozygous invalid mutation in the IL2rg gene; and iii. implantation of human hematopoietic progenitor cells, and b. determining whether the agent reduces the toxicity of the toxic drug to human red blood cells in the non-human animal.

[0369] In exemplary embodiment 52, provided herein is the method of embodiment 51, wherein the agent and the toxic drug are administered to the non-human animal simultaneously or sequentially.

[0370] In exemplary embodiment 53, provided herein is the method of any one of embodiments 37-52, wherein the transgenic non-human animal comprises a homozygous null mutation in the Rag1 gene.

[0371] In exemplary embodiment 54, provided herein is a method of any one of embodiments 37-53, wherein the null mutation is a deletion of at least exons corresponding to mouse Hmox-1 exons 3-5.

[0372] In exemplary embodiment 55, provided herein is the method of any one of embodiments 37-54, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0373] In exemplary embodiment 56a, provided herein is the method of any one of embodiments 37-55, wherein the transgenic non-human animal comprises an implant of human hepatocytes.

[0374] In exemplary embodiment 56b, provided herein is the method of any one of embodiments 37-56a, wherein the transgenic non-human animal comprises a homozygous null mutation in the Fah gene.

[0375] In exemplary embodiment 57, provided herein is the method of embodiment 56b, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0376] In exemplary embodiment 58, provided herein is the method of any one of embodiments 37-57, wherein the transgenic non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0377] In exemplary embodiment 59, provided herein is the method of embodiment 58, wherein the transgenic non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0378] In exemplary embodiment 60, provided herein is the method of embodiment 59, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0379] In exemplary embodiment 61, provided herein is the method of embodiment 59 or 60, wherein the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

[0380] In exemplary embodiment 62, provided herein is a method of any one of embodiments 59-61, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0381] In exemplary embodiment 63, provided herein is the method of embodiment 58, wherein the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0382] In exemplary embodiment 64, provided herein is a method of any one of embodiments 58-63, wherein the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0383] In exemplary embodiment 65, provided herein is a method of any one of embodiments 58-64, wherein at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

[0384] In exemplary embodiment 66, provided herein is the method of embodiment 65, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0385] In exemplary embodiment 67, provided herein is the method of embodiment 65 or 66, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0386] In exemplary embodiment 68, provided herein is the method of any one of embodiments 58-67, wherein the transgenic non-human animal comprises a null mutation in at least one corresponding non-human animal gene located at the corresponding non-human animal locus.

[0387] In exemplary embodiment 69, provided herein is a method of any one of embodiments 58-68, wherein the transgenic non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0388] In exemplary embodiment 70, provided herein is a method of any one of embodiments 58-69, wherein the transgenic non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0389] In exemplary embodiment 71, provided herein is the method of any one of embodiments 58-70, wherein the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0390] In exemplary embodiment 72, provided herein is the method of any one of embodiments 58-71, wherein the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0391] In exemplary embodiment 73, provided herein is the method of embodiment 72, wherein the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0392] In exemplary embodiment 74, provided herein is a method of any one of embodiments 58-73, wherein the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0393] In exemplary embodiment 75, provided herein is a method of any one of embodiments 58-74, wherein the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0394] In exemplary embodiment 76, provided herein is the method of any one of embodiments 37-75, wherein the transgenic non-human animal is a mammal.

[0395] In exemplary embodiment 77, provided herein is the method of embodiment 76, wherein the mammal is a rodent, such as a rat or a mouse.

[0396] In exemplary embodiment 78, provided herein is the method of embodiment 77, wherein the rodent is a mouse.

[0397] In exemplary embodiment 79, provided herein is a transgenic non-human animal cell comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

[0398] In exemplary embodiment 80, provided herein is the transgenic non-human animal cell of embodiment 79, comprising a homozygous null mutation in the Rag1 gene.

[0399] In exemplary embodiment 81, provided herein is the transgenic non-human animal cell of embodiment 79 or 80, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

[0400] In exemplary embodiment 82, provided herein is the transgenic non-human animal cell of any one of embodiments 79-81, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0401] In exemplary embodiment 83, provided herein is the transgenic non-human animal cell of any one of embodiments 79-82, comprising a homozygous null mutation in the Fah gene.

[0402] In exemplary embodiment 84, provided herein is the transgenic non-human animal cell of embodiment 83, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0403] In exemplary embodiment 85, provided herein is the transgenic non-human animal cell of any one of embodiments 79-84, wherein the transgenic non-human animal cell expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0404] In exemplary embodiment 86, provided herein is the transgenic non-human animal cell of embodiment 85, wherein the transgenic non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide, wherein the Sirpa polypeptide comprises an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0405] In exemplary embodiment 87, provided herein is the transgenic non-human animal cell of embodiment 86, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0406] In exemplary embodiment 88, provided herein is the transgenic non-human animal cell of embodiment 86 or 87, wherein the transgenic non-human animal cell expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

[0407] In exemplary embodiment 89, provided herein is the transgenic non-human animal cell of any one of embodiments 86-88, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0408] In exemplary embodiment 90, provided herein is the transgenic non-human animal cell of embodiment 85, wherein the transgenic non-human animal cell expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0409] In exemplary embodiment 91, provided herein is a transgenic non-human animal cell according to any one of embodiments 85-90, wherein the transgenic non-human animal cell further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0410] In exemplary embodiment 92, provided herein is a transgenic non-human animal cell of any one of embodiments 85-91, wherein at least one promoter operably linked to the nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0411] In exemplary embodiment 93, provided herein is the transgenic non-human animal cell of embodiment 92, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0412] In exemplary embodiment 94, provided herein is the transgenic non-human animal cell of embodiment 92 or 93, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0413] In exemplary embodiment 95, provided herein is a transgenic non-human animal cell of any one of embodiments 85-94, comprising a null mutation in at least one corresponding non-human animal gene located at the corresponding non-human animal locus.

[0414] In exemplary embodiment 96, provided herein is a transgenic non-human animal cell of any one of embodiments 85-95, wherein the transgenic non-human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0415] In exemplary embodiment 97, provided herein is a transgenic non-human animal cell of any one of embodiments 85-95, wherein the transgenic non-human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0416] In exemplary embodiment 98, provided herein is the transgenic non-human animal cell of any one of embodiments 85-97, wherein the transgenic non-human animal cell expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0417] In exemplary embodiment 99, provided herein is a transgenic non-human animal cell of any one of embodiments 85-98, wherein the transgenic non-human animal cell expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0418] In exemplary embodiment 100, provided herein is the transgenic non-human animal cell of embodiment 99, wherein the transgenic non-human animal cell expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0419] In exemplary embodiment 101, provided herein is a transgenic non-human animal cell according to any one of embodiments 85-100, wherein the transgenic non-human animal cell expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0420] In exemplary embodiment 102, provided herein is a transgenic non-human animal cell of any one of embodiments 85-101, wherein the transgenic non-human animal cell expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0421] In exemplary embodiment 103, provided herein is the transgenic non-human animal cell of any one of embodiments 79-102, wherein the transgenic non-human animal cell is a mammalian cell.

[0422] In exemplary embodiment 104, provided herein is the transgenic non-human animal cell of embodiment 103, wherein the mammalian cell is a rodent cell, such as a rat cell or a mouse cell.

[0423] In exemplary embodiment 105, provided herein is the transgenic non-human animal cell of embodiment 104, wherein the rodent cell is a mouse cell.

[0424] In exemplary embodiment 106, provided herein is a transgenic non-human animal cell of any one of embodiments 79-84, 86-87, 89, 92-97, and 103-105, wherein the transgenic non-human animal cell is a non-human animal embryonic stem (ES) cell.

[0425] In exemplary embodiment 107, the present invention provides a non-human animal embryonic stem cell, comprising in its genome: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0426] In exemplary embodiment 108, provided herein is the non-human animal embryonic stem cell of embodiment 107, wherein the non-human animal embryonic stem cell comprises a homozygous null mutation in the Rag1 gene in its genome.

[0427] In exemplary embodiment 109, provided herein is the non-human animal embryonic stem cell of embodiment 107 or 108, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

[0428] In exemplary embodiment 110, provided herein is the non-human animal embryonic stem cell of any one of embodiments 107-109, wherein the null mutation is a deletion of the entire endogenous coding sequence of Hmox-1.

[0429] In exemplary embodiment 111, provided herein is the non-human animal embryonic stem cell of any one of embodiments 107-110, wherein the non-human animal embryonic stem cell comprises a homozygous null mutation in the Fah gene.

[0430] In exemplary embodiment 112, provided herein is the non-human animal embryonic stem cell of embodiment 111, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0431] In exemplary embodiment 113, provided herein is the non-human animal embryonic stem cell of any one of embodiments 107-112, wherein the non-human animal embryonic stem cell comprises in its genome a nucleic acid encoding a human or humanized SIRPA polypeptide, and wherein the nucleic acid is operably linked to a Sirpa promoter.

[0432] In exemplary embodiment 114, provided herein is the non-human animal embryonic stem cell of embodiment 113, wherein the transgenic non-human animal embryonic stem cell comprises a Sirpa gene encoding a Sirpa polypeptide, wherein the Sirpa polypeptide comprises an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0433] In exemplary embodiment 115, provided herein is the non-human animal embryonic stem cell of embodiment 114, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0434] In exemplary embodiment 116, provided herein is the non-human animal embryonic stem cell of any one of embodiments 114-115, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide.

[0435] In exemplary embodiment 117, provided herein is the non-human animal embryonic stem cell of any one of embodiments 114-115, wherein the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0436] In exemplary embodiment 118, provided herein is the non-human animal embryonic stem cell of embodiment 113, wherein the transgenic non-human animal embryonic stem cell comprises a nucleic acid encoding a Sirpa polypeptide, and the nucleic acid is operably linked to a Sirpa promoter.

[0437] In exemplary embodiment 119, provided herein is a non-human animal embryonic stem cell according to any one of embodiments 113-118, wherein the transgenic non-human animal embryonic stem cell further comprises in its genome one or more nucleic acids selected from the group consisting of: a nucleic acid encoding a human TPO protein and operably linked to a TPO promoter; a nucleic acid encoding a human GM-CSF protein and operably linked to a GM-CSF promoter; a nucleic acid encoding a human IL3 protein and operably linked to an IL3 promoter; a nucleic acid encoding a human IL15 protein and operably linked to an IL15 promoter; a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter; and a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0438] In exemplary embodiment 120, provided herein is a non-human animal embryonic stem cell of any one of embodiments 113-119, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0439] In exemplary embodiment 121, provided herein is the non-human animal embryonic stem cell of embodiment 120, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0440] In exemplary embodiment 122, provided herein is the non-human animal embryonic stem cell of embodiment 120 or 121, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0441] In exemplary embodiment 123, provided herein is the non-human animal embryonic stem cell of any one of embodiments 113-122, comprising a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

[0442] In exemplary embodiment 124, provided herein is a non-human animal embryonic stem cell of any one of embodiments 113-123, wherein the transgenic non-human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0443] In exemplary embodiment 125, provided herein is a non-human animal embryonic stem cell of any one of embodiments 113-123, wherein the transgenic non-human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0444] In exemplary embodiment 126, provided herein is the non-human animal embryonic stem cell of any one of embodiments 113-125, wherein the transgenic non-human animal embryonic stem cell comprises in its genome a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter.

[0445] In exemplary embodiment 127, provided herein is the non-human animal embryonic stem cell of any one of embodiments 113-126, wherein the transgenic non-human animal embryonic stem cell comprises in its genome a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0446] In exemplary embodiment 128, provided herein is the non-human animal embryonic stem cell of embodiment 127, wherein the transgenic non-human animal embryonic stem cell comprises in its genome a nucleic acid encoding a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0447] In exemplary embodiment 129, provided herein is the non-human animal embryonic stem cell of any one of embodiments 113-128, wherein the transgenic non-human animal embryonic stem cell comprises in its genome: (i) a nucleic acid encoding a human or humanized SIRPA protein and operably linked to a Sirpa promoter; (ii) a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; and (iii) a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0448] In exemplary embodiment 130, provided herein is the non-human animal embryonic stem cell of any one of embodiments 113-129, wherein the transgenic non-human animal embryonic stem cell comprises in its genome a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0449] In exemplary embodiment 131, provided herein is the non-human animal embryonic stem cell of any one of embodiments 107-130, wherein the transgenic non-human animal embryonic stem cell is a mammalian embryonic stem cell.

[0450] In exemplary embodiment 132, provided herein is the non-human animal embryonic stem cell of embodiment 131, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell, such as a rat embryonic stem cell or a mouse embryonic stem cell.

[0451] In exemplary embodiment 133, provided herein is the non-human animal embryonic stem cell of embodiment 132, wherein the rodent embryonic stem cell is a mouse embryonic stem cell.

[0452] In exemplary embodiment 134, the present invention provides a method for producing non-human animal embryonic stem cells, comprising genetically engineering the non-human animal embryonic stem cells so that the non-human animal embryonic stem cells have a genome comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0453] In exemplary embodiment 135, provided herein is the method of embodiment 134, wherein the non-human animal embryonic stem cell is further engineered to contain a homozygous null mutation in the Rag1 gene in its genome.

[0454] In exemplary embodiment 136, provided herein is the method of embodiment 134 or 135, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

[0455] In exemplary embodiment 137, provided herein is the method of any one of embodiments 134-136, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0456] In exemplary embodiment 138, provided herein is a method of any one of embodiments 134-137, wherein the non-human animal embryonic stem cell is further engineered to comprise a homozygous null mutation in the Fah gene in its genome.

[0457] In exemplary embodiment 139, provided herein is the method of embodiment 138, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0458] In exemplary embodiment 140, provided herein is a method of any one of embodiments 134-139, wherein the non-human animal embryonic stem cell is further engineered to contain in its genome a nucleic acid encoding a human or humanized SIRPA polypeptide, and wherein the nucleic acid is operably linked to a Sirpa promoter.

[0459] In exemplary embodiment 141, provided herein is a method of embodiment 140, wherein the transgenic non-human animal embryonic stem cell comprises a Sirpa gene encoding a Sirpa polypeptide, wherein the Sirpa polypeptide comprises an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0460] In exemplary embodiment 142, provided herein is the method of embodiment 141, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0461] In exemplary embodiment 143, provided herein is the method of embodiment 141 or 142, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide.

[0462] In exemplary embodiment 144, provided herein is the method of any one of embodiments 141 or 142, wherein the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0463] In exemplary embodiment 145, provided herein is the method of embodiment 140, wherein the transgenic non-human animal embryonic stem cell comprises a nucleic acid encoding a human SIRPA polypeptide, and the nucleic acid is operably linked to a Sirpa promoter.

[0464] In exemplary embodiment 146, provided herein is a method of any one of embodiments 140-145, wherein the transgenic non-human animal embryonic stem cell is further engineered to contain in its genome one or more nucleic acids selected from the group consisting of: a nucleic acid encoding a human TPO protein and operably linked to a TPO promoter; a nucleic acid encoding a human GM-CSF protein and operably linked to a GM-CSF promoter; a nucleic acid encoding a human IL3 protein and operably linked to an IL3 promoter; a nucleic acid encoding a human IL15 protein and operably linked to an IL15 promoter; a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter; and a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0465] In exemplary embodiment 147, provided herein is a method of any one of embodiments 140-146, wherein at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

[0466] In exemplary embodiment 148, provided herein is the method of embodiment 147, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0467] In exemplary embodiment 149, provided herein is the method of embodiment 147 or 148, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0468] In exemplary embodiment 150, provided herein is a method of any one of embodiments 140-149, comprising a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

[0469] In exemplary embodiment 151, provided herein is a method of any one of embodiments 140-150, wherein the transgenic non-human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0470] In exemplary embodiment 152, provided herein is a method of any one of embodiments 140-150, wherein the transgenic non-human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0471] In exemplary embodiment 153, provided herein is a method of any one of embodiments 140-152, wherein the transgenic non-human animal embryonic stem cell is engineered to contain in its genome a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter.

[0472] In exemplary embodiment 154, provided herein is a method of any one of embodiments 140-153, wherein the transgenic non-human animal embryonic stem cell is engineered to contain in its genome a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0473] In exemplary embodiment 155, provided herein is a method of embodiment 154, wherein the transgenic non-human animal cell embryonic stem cell is engineered to contain a nucleic acid encoding a humanized CD47 protein in its genome, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0474] In exemplary embodiment 156, provided herein is a method of any one of embodiments 140-155, wherein the transgenic non-human animal embryonic stem cell is engineered to contain in its genome: (i) a nucleic acid encoding a human or humanized SIRPA protein and operably linked to a Sirpa promoter; (ii) a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; and (iii) a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0475] In exemplary embodiment 157, provided herein is a method of any one of embodiments 140-156, wherein the transgenic non-human animal embryonic stem cell is engineered to contain in its genome a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0476] In exemplary embodiment 158, provided herein is a method of any one of embodiments 134-157, wherein the transgenic non-human animal embryonic stem cell is a mammalian embryonic stem cell.

[0477] In exemplary embodiment 159, provided herein is the method of embodiment 158, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell, such as a rat embryonic stem cell or a mouse embryonic stem cell.

[0478] In exemplary embodiment 160, provided herein is the method of embodiment 159, wherein the rodent embryonic stem cells are mouse embryonic stem cells.

[0479] In exemplary embodiment 161, provided herein is a non-human animal embryo, comprising the non-human animal embryonic stem cell described in any one of embodiments 106-133, or a non-human animal embryonic stem cell produced according to the method described in any one of embodiments 134-160.

[0480] In exemplary embodiment 162, provided herein is a method for producing a non-human animal, the genome of which contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising the following steps: (a) obtaining a non-human animal embryonic stem cell according to any one of embodiments 106-133, or a non-human animal embryonic stem cell made according to the method of any one of embodiments 134-160; and (b) creating a non-human animal using the non-human animal embryonic cells of (a).

[0481] In exemplary embodiment 163, the present invention provides a method for producing a non-human animal, the genome of which contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising modifying the genome of the non-human animal so that it contains: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

[0482] In exemplary embodiment 164, provided herein is the method of embodiment 163, wherein the transgenic non-human animal is further engineered to comprise a homozygous null mutation in the Rag1 gene.

[0483] In exemplary embodiment 165, provided herein is the method of embodiment 163 or 164, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

[0484] In exemplary embodiment 166, provided herein is the method of any one of embodiments 163-165, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0485] In exemplary embodiment 167, provided herein is the method of any one of embodiments 163-166, wherein the transgenic non-human animal is further engineered to comprise a homozygous null mutation in the Fah gene.

[0486] In exemplary embodiment 168, provided herein is the method of embodiment 167, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

[0487] In exemplary embodiment 169, provided herein is the method of any one of embodiments 163-168, wherein the transgenic non-human animal is further engineered to express a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0488] In exemplary embodiment 170, provided herein is the method of embodiment 169, wherein the transgenic non-human animal is engineered to comprise a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0489] In exemplary embodiment 171, provided herein is the method of embodiment 170, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0490] In exemplary embodiment 172, provided herein is the method of embodiment 170 or 171, wherein the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

[0491] In exemplary embodiment 173, provided herein is a method of any one of embodiments 170-172, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0492] In exemplary embodiment 174, provided herein is the method of embodiment 169, wherein the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0493] In exemplary embodiment 175, provided herein is a method of any one of embodiments 169-174, wherein the transgenic non-human animal is further engineered to express one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0494] In exemplary embodiment 176, provided herein is a method of any one of embodiments 169-175, wherein at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

[0495] In exemplary embodiment 177, provided herein is the method of embodiment 176, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

[0496] In exemplary embodiment 178, provided herein is the method of embodiment 176 or 177, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

[0497] In exemplary embodiment 179, provided herein is a method of any one of embodiments 169-178, comprising a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

[0498] In exemplary embodiment 180, provided herein is a method of any one of embodiments 169-179, wherein the transgenic non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0499] In exemplary embodiment 181, provided herein is a method of any one of embodiments 169-180, wherein the transgenic non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

[0500] In exemplary embodiment 182, provided herein is the method of any one of embodiments 169-181, wherein the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0501] In exemplary embodiment 183, provided herein is the method of any one of embodiments 169-182, wherein the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0502] In exemplary embodiment 184, provided herein is the method of embodiment 183, wherein the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0503] In exemplary embodiment 185, provided herein is a method of any one of embodiments 169-184, wherein the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0504] In exemplary embodiment 186, provided herein is a method of any one of embodiments 169-185, wherein the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0505] In exemplary embodiment 187, provided herein is the method of any one of embodiments 163-186, wherein the transgenic non-human animal is a mammal.

[0506] In exemplary embodiment 188, provided herein is the method of embodiment 187, wherein the mammal is a rodent, such as a rat or a mouse.

[0507] In exemplary embodiment 189, provided herein is the method of embodiment 188, wherein the rodent is a mouse. Example

[0508] The following examples and drawings are provided to describe how to make and use the methods and compositions of the present invention to those of ordinary skill in the art, and are not intended to limit the scope of the inventor's invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be considered. These examples do not include a detailed description of conventional methods (molecular cloning techniques, etc.) known to those of ordinary skill in the art. Unless otherwise stated, parts are weight parts, molecular weights are average molecular weights, temperatures are expressed in degrees Celsius, and pressures are equal to or close to atmospheric pressure.

[0509] Example 1: Generation of Hmox-1 knockout mice

[0510] use The mouse Hmox-1 gene was deleted in the mouse genome by technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659, both of which are incorporated herein by reference). On mouse chromosome 8 C1, the 7 kb mouse genomic sequence of the mouse Hmox-1 gene from the start codon ATG to the stop codon between coordinates chr8: 75093750-75100019 (GRCm38 assembly) was deleted. Figure 1 .

[0511] In detail, mouse homology arms were prepared by PCR amplification using BAC clone RP23-102I24 as template and are shown in Table 1 below:

[0512] Table 1

[0513]

[0514] To prepare a targeting vector (designated MAID20433) from mouse BAC clone RP23-102I24, a hygromycin (Hyg) resistance self-deletion cassette (with CRE recombinase controlled by a protamine promoter) flanked by mutated lox sites (lox2372-hyg-lox2372) was used to replace the ~7 kb mouse sequence containing the mouse Hmox-1 gene by bacterial homologous recombination (BHR).

[0515] The final targeting vector contains from 5' to 3': a chloramphenicol resistance cassette (CM; Figure 1 not depicted above), 5' mouse homology arm, lox2372-Hyg-lox2372 self-deletion cassette, 3' mouse homology arm; final clones were selected based on CM / Hyg resistance.

[0516] The MAID20433 targeting vector was electroporated into mouse embryonic stem (ES) cells. Targeted homologous recombination resulted in ~7kb mouse sequence deletion (GRCm38 coordinates chr8:75093750-75100019). As described above, successful integration was confirmed by modified allele (MOA) determination, for example, see Valenzuela et al., supra. Primers and probes for MOA determination of mouse Hmox-1 sequence loss are shown in Table 2 below. The cassette was subsequently removed by expressing CRE recombinase (controlled by protamine promoter) in mice.

[0517] Table 2

[0518]

[0519] Positively targeted ES cells were used as donor ES cells and Methods Microinjection into pre-morula (8-cell) stage mouse embryos (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1, all of which are incorporated herein by reference). Mouse embryos containing donor ES cells are incubated in vitro and then implanted into surrogate mothers to produce F0 mice derived entirely from donor ES cells. Mice with Hmox-1 gene deletion are identified by genotyping using the above-mentioned MOA assay. Hmox-1 gene deletion heterozygous mice are bred to homozygosity. HMOX-1KO mice also include Rag2 knockout, Il2rg knockout, human M-CSF, humanized Sripa and humanized CD47 genes (described in WO 2011 / 044050, WO 2012 / 112544, WO 2014 / 039782, WO 2014 / 071397, US Pat. No. 11,019,810, WO 2014 / 039782, WO 2014 / 071397, WO 2016 / 168212, U.S. Pat. No. 10,918,095, WO 2018 / 177440 and WO 2016 / 089692, each of which is incorporated herein by reference), which are obtained by ES cell modification or breeding. Mice are bred to be homozygous for all genes. Throughout the examples, these mice are designated as HMOX-1 - / - HIS or HMOX-1 - / - MSRG47 mice. Mice were maintained on a sulfonamide diet (LabDiet, St. Louis, MO) at the MPF facility and bred in-house for approximately 5-10 generations.

[0520] Example 2: Hmox-1 deficiency in mice reduces mouse macrophages

[0521] To evaluate whether the loss of Hmox-1 gene has an effect on mouse macrophages, HMOX-1 - / -MSRG47 mouse spleens were harvested into RPMI containing 10% fetal bovine serum (FBS) and single cell suspensions were prepared by mechanical digestion and then passed through a 70 mm mesh filter. Blood was collected by cardiac puncture into PBS containing 5 mM EDTA. Blood and spleen cells were treated with ACK lysis buffer (Gibco) to lyse red blood cells and then resuspended in PBS containing 2% FBS and 1 mM EDTA and human and mouse Fc blocker (BD Biosciences). Cells were counted and then stained with anti-mouse CD45 APC-Cy7 (clone 30-F11; BD Biosciences), CD14 FITC (clone Sa14-2; Biolegend), CD11b BV605 (clone M1 / 70; Biolegend), F4 / 80 PE-Dazzle (clone BM8; Biolegend), VCAM PE (clone 429; Biolegend), and anti-human CD45 PE-Cy5.5 (clone HI30; Invitrogen). Cells were washed three times and then acquired on a BD FACSsymphony. FlowJo 10.7 software was used for analysis, and hCD45 - / mCD45 + Cells were gated on the population and then analyzed for mouse monocytes (CD11b + / CD14 + ) and mouse macrophages (CD11b + / CD14 + F4 / 80 + ).

[0522] like Figure 2A -B, HMOX-1 - / - MSRG47 mice have a reduction in mouse macrophages, but not monocytes, in the blood and spleen. Thus, the primary cells responsible for clearing human erythrocytes (mouse macrophages) are reduced, thereby improving the survival of human erythrocytes introduced into the body via hematopoietic stem cell (HSC) implantation or passive transfusion. In addition, by reducing mouse macrophages but still maintaining mouse myeloid cells, this mouse strain does not develop other pathological conditions caused by the complete loss of myeloid cells (e.g., mouse CSF-1R - / - ; Dai et al. 2002. Blood. 99: 111-120).

[0523] Example 3: Effects of Hmox-1 deficiency on human erythrocytes

[0524] To evaluate the effect of Hmox-1 deficiency on human erythrocytes, human erythrocytes were first prepared from human buffy coats (BioIVT) diluted 2-fold in PBS containing 5 mM EDTA. 400 million erythrocytes were injected intraperitoneally into untransplanted HMOX-1 + / + and HMOX-1 - / - MSRG47 mice. Mice were bled retro-orbitally on days 1, 2, 6, and 8 after injection, and peripheral blood was collected in PBS containing 5 mM EDTA and analyzed by FACS staining with anti-human CD235a (human erythrocyte marker; clone HI264, Biolegend) and anti-mouse Ter119 PE-Cy7 (mouse erythrocyte marker; clone TER-119; Biolegend). Cells were collected on a BD FACS Symphony and stained for C...

Claims

1. A transgenic non-human animal comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

2. The transgenic non-human animal according to claim 1, comprising a homozygous null mutation in the Rag1 gene.

3. The transgenic non-human animal according to claim 1 or 2, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

4. The transgenic non-human animal according to any one of claims 1 to 3, wherein the null mutation is a deletion of the entire endogenous coding sequence of Hmox-1.

5. The transgenic non-human animal according to any one of claims 1 to 4, comprising a homozygous null mutation in the Fah gene.

6. The transgenic non-human animal according to claim 5, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

7. The transgenic non-human animal of any one of claims 1-6, wherein the transgenic non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

8. The transgenic non-human animal of claim 7, wherein the transgenic non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

9. The transgenic non-human animal of claim 8, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

10. The transgenic non-human animal of claim 8 or 9, wherein the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

11. The transgenic non-human animal according to any one of claims 8-10, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

12. The transgenic non-human animal of claim 7, wherein the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

13. The transgenic non-human animal of any one of claims 7-12, wherein the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

14. The transgenic non-human animal of any one of claims 7-13, wherein at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

15. The transgenic non-human animal of claim 14, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

16. A transgenic non-human animal according to claim 14 or 15, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

17. The transgenic non-human animal of any one of claims 7-16, comprising a null mutation in at least one corresponding non-human animal gene at the corresponding non-human animal locus.

18. The transgenic non-human animal of any one of claims 7-17, wherein the transgenic non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

19. The transgenic non-human animal of any one of claims 7-17, wherein the transgenic non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

20. The transgenic non-human animal of any one of claims 7-19, wherein the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

21. The transgenic non-human animal of any one of claims 7-20, wherein the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

22. The transgenic non-human animal of claim 21, wherein the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

23. The transgenic non-human animal of any one of claims 7-22, wherein the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

24. The transgenic non-human animal of any one of claims 7-23, wherein the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

25. The transgenic non-human animal of any one of claims 1-24, further comprising an implant of human hematopoietic cells.

26. The transgenic non-human animal according to claim 25, wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human erythroid precursor cells, and human erythrocytes.

27. The transgenic non-human animal of claim 36, wherein the animal comprises human erythroid cells.

28. The transgenic non-human animal of claim 27, wherein the non-human animal further comprises an infection with a pathogen that targets human erythroid cells.

29. The transgenic non-human animal of any one of claims 25-28, wherein the animal comprises an inactivated endogenous FAH gene, and wherein the animal further comprises transplanted human hepatocytes.

30. The transgenic non-human animal of claim 28 or claim 29, wherein the pathogen is capable of causing malaria in humans.

31. The transgenic non-human animal of claim 28, wherein the pathogen is selected from the group consisting of Plasmodium sp., Babesia sp., and Theileri sp.

32. The transgenic non-human animal of any one of claims 25-27, wherein the implanted human hematopoietic cells produce abnormal human erythroid cells.

33. The transgenic non-human animal of claim 32, wherein the implanted human hematopoietic cells comprise a mutation in the beta-globin gene that causes sickle cell disease.

34. The transgenic non-human animal of any one of claims 1-33, wherein the transgenic non-human animal is a mammal.

35. The transgenic non-human animal of claim 34, wherein the mammal is a rodent, such as a rat or a mouse.

36. The transgenic non-human animal of claim 35, wherein the rodent is a mouse.

37. A method for identifying an agent that inhibits infection by a pathogen that targets human erythroid cells, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; iii. implantation of artificial hematopoietic cells; and iv. infection with a pathogen that targets human erythroid cells, and b. Determining whether the agent reduces the amount of and / or inhibits the activity of a pathogen in a non-human animal infected with the pathogen.

38. A method for identifying an agent that prevents infection by a pathogen that targets human erythroid cells, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. Implantation of artificial hematopoietic cells, b. injecting parasite-infected reticulocytes or erythrocytes into the transgenic non-human animal, and c. Determining whether the agent prevents infection of a non-human animal by the human reticulocytes and / or the red blood cells.

39. The method of claim 37 or 38, wherein the pathogen is capable of causing malaria in humans.

40. The method of claim 37 or 38, wherein the pathogen is selected from Plasmodium species, Babesia species, and Theileria species.

41. A method of identifying an agent for treating sickle cell disease, the method comprising: a. administering the agent to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. implantation of human hematopoietic cells comprising a β-globin gene mutation that causes sickle cell disease, and b. Determining whether the agent prevents or reduces red blood cell sickling in the non-human animal.

42. A method for evaluating the therapeutic effect of a candidate drug targeting human red blood cells, the method comprising: a. administering the candidate drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. Engraftment of human hematopoietic progenitor cells, and b. monitoring the human red blood cells in the non-human animal to evaluate the therapeutic effect of the candidate drug.

43. The method of claim 42, wherein the human red blood cells are monitored to determine whether the candidate drug increases the production and / or survival of human red blood cells in the non-human animal.

44. A method for evaluating the toxicity of a candidate drug to human red blood cells, comprising: a. administering the candidate drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. Engraftment of human hematopoietic progenitor cells, and b. monitoring the human red blood cells in the non-human animal to assess the toxicity of the drug candidate.

45. The method of claim 44, wherein the toxicity is on-target toxicity or off-target toxicity.

46. ​​The method of claim 44 or 45, wherein the human red blood cells are monitored to determine whether the candidate drug reduces the number of human red blood cells in the non-human animal.

47. The method of claim 46, wherein the candidate drug is a chemotherapeutic agent or an anti-malarial agent.

48. The method of claim 44 or 45, wherein the human red blood cells are monitored to assess whether the candidate drug induces agglutination of the red blood cells.

49. The method of claim 48, wherein the candidate drug is a modulator of human CD47 protein.

50. The method of claim 49, wherein the modulator is an antibody.

51. A method for identifying an agent that reduces the toxicity of a toxic drug to human red blood cells, comprising: a. administering the agent and the toxic drug to a transgenic non-human animal, wherein the transgenic non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. Engraftment of human hematopoietic progenitor cells, and b. Determining whether the agent reduces the toxicity of the toxic drug to human red blood cells in the non-human animal.

52. The method of claim 51, wherein the agent and the toxic drug are administered to the non-human animal simultaneously or sequentially.

53. The method of any one of claims 37-52, wherein the transgenic non-human animal comprises a homozygous null mutation in the Rag1 gene.

54. The method of any one of claims 37-53, wherein the null mutation is a deletion of at least exons corresponding to mouse Hmox-1 exons 3-5.

55. The method of any one of claims 37-54, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

56. The method of any one of claims 37-55, comprising a homozygous null mutation in the Fah gene.

57. The method of claim 56, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in the endogenous Fah gene.

58. The method of any one of claims 37-57, wherein the transgenic non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

59. The method of claim 58, wherein the transgenic non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

60. The method of claim 59, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

61. The method of claim 59 or 60, wherein the transgenic non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

62. The method of any one of claims 59-61, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

63. The method of claim 58, wherein the transgenic non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

64. The method of any one of claims 58-63, wherein the transgenic non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

65. The method of any one of claims 58-64, wherein at least one promoter operably linked to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter.

66. The method of claim 65, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

67. A method according to claim 65 or 66, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

68. The method of any one of claims 58-67, comprising an invalid mutation in at least one corresponding non-human animal gene located at the corresponding non-human animal locus.

69. The method of any one of claims 58-68, wherein the transgenic non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

70. The method of any one of claims 58-69, wherein the transgenic non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

71. The method of any one of claims 58-70, wherein the transgenic non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

72. The method of any one of claims 58-71, wherein the transgenic non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

73. The method of claim 72, wherein the transgenic non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

74. The method of any one of claims 58-73, wherein the transgenic non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

75. The method of any one of claims 58-74, wherein the transgenic non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

76. The method of any one of claims 37-75, wherein the transgenic non-human animal is a mammal.

77. The method of claim 76, wherein the mammal is a rodent, such as a rat or a mouse.

78. The method of claim 77, wherein the rodent is a mouse.

79. A transgenic non-human animal cell comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

80. The transgenic non-human animal cell of claim 79, comprising a homozygous null mutation in the Rag1 gene.

81. The transgenic non-human animal cell of claim 79 or 80, wherein the null mutation is a deletion of at least exons corresponding to exons 3-5 of mouse Hmox-1.

82. The transgenic non-human animal cell of any one of claims 79-81, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

83. The transgenic non-human animal cell of any one of claims 79-82, comprising a homozygous null mutation in the Fah gene.

84. The transgenic non-human animal cell of claim 83, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion and / or substitution in an endogenous Fah gene.

85. The transgenic non-human animal cell of any one of claims 79-84, wherein the transgenic non-human animal cell expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

86. The transgenic non-human animal cell of claim 85, wherein the transgenic non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

87. The transgenic non-human animal cell of claim 86, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

88. The transgenic non-human animal cell of claim 86 or 87, wherein the transgenic non-human animal cell expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

89. The transgenic non-human animal cell of any one of claims 86-88, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

90. The transgenic non-human animal cell of claim 85, wherein the transgenic non-human animal cell expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

91. The transgenic non-human animal cell of any one of claims 85-90, wherein the transgenic non-human animal cell further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

92. The transgenic non-human animal cell of any one of claims 85-91, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

93. A transgenic non-human animal cell according to claim 92, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

94. A transgenic non-human animal cell according to claim 92 or 93, wherein the endogenous non-human animal promoter is located at the corresponding non-human animal locus.

95. The transgenic non-human animal cell of any one of claims 85-94, comprising an invalid mutation in at least one corresponding non-human animal gene located at the corresponding non-human animal locus.

96. A transgenic non-human animal cell according to any one of claims 85-95, wherein the transgenic non-human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

97. A transgenic non-human animal cell according to any one of claims 85-95, wherein the transgenic non-human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding the human or humanized protein.

98. The transgenic non-human animal cell of any one of claims 85-97, wherein the transgenic non-human animal cell expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

99. The transgenic non-human animal cell of any one of claims 85-98, wherein the transgenic non-human animal cell expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

100. The transgenic non-human animal cell of claim 99, wherein the transgenic non-human animal cell expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

101. The transgenic non-human animal cell of any one of claims 85-100, wherein the transgenic non-human animal cell expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to a M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

102. A transgenic non-human animal cell according to any one of claims 85-101, wherein the transgenic non-human animal cell expresses human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

103. The transgenic non-human animal cell of any one of claims 79-102, wherein the transgenic non-human animal cell is a mammalian cell.

104. The transgenic non-human animal cell of claim 103, wherein the mammalian cell is a rodent cell, such as a rat cell or a mouse cell.

105. The transgenic non-human animal cell of claim 104, wherein the rodent cell is a mouse cell.

106. The transgenic non-human animal cell of any one of claims 79-84, 86-87, 89, 92-97 and 103-105, wherein the transgenic non-human animal cell is a non-human animal embryonic stem cell (ES).

107. A method for producing non-human animal embryonic stem cells, comprising genetically engineering the non-human animal embryonic stem cells so that the non-human animal embryonic stem cells have a genome comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

108. A non-human animal embryo, comprising the non-human animal embryonic stem cells according to claim 106, or the non-human animal embryonic stem cells produced according to the method of claim 107.

109. A method for producing a non-human animal, wherein the genome of the non-human animal comprises: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising the following steps: (a) obtaining the non-human animal embryonic stem cells according to claim 106, or the non-human animal embryonic stem cells prepared according to the method of claim 107; and (b) Creating a non-human animal using the non-human animal embryonic cell of (a).

110. A method for producing a non-human animal, wherein the genome of the non-human animal comprises: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal, the method comprising modifying the genome of the non-human animal so that it comprises: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the heme oxygenase-1 (Hmox-1) gene of the non-human animal.

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